Human Amniotic Membrane

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Aug 8, 2013 - Vaginoplasty. - Conjunctival reconstruction. - Treatment of burns and traumatic skin wounds. - Intra-abdominal treatment of adhesion. Trelford ...
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Human Amniotic Membrane: Clinical Uses, Patents and Marketed Products Florelle Gindraux1,2,3*, Romain Laurent1,4, Laurence Nicod1,5, Benoit de Billy1,4, Christophe Meyer1,6, Narcisse Zwetyenga1,7, Luc Wajszczak1,7, Patrick Garbuio1,2 and Laurent Obert1,2 1

Intervention, Innovation, Imagery, Engineering in Health (EA 4268), SFR FED 4234, University of Franche-Comté, Besancon, France; 2Orthopaedic and Traumatology Surgery Service, University Hospital of Besancon, France; 3 Clinical Investigation Centre in Biotherapy, University Hospital of Besancon, France; 4Paediatric Surgery Service, University Hospital of Besancon, France; 5 Cell Biology Laboratory, Faculty of Medicine-Pharmacy, Besancon, France; 6 Maxillofacial Surgery Service, University Hospital of Besancon, France; 7Department of Maxillofacial Surgery, Plastic - Reconstructive and Aesthetic Surgery, Hand Surgery, University Hospital of Dijon, France Received: June 26, 2013; Accepted: August 8, 2013; Revised: August 8, 2013

Abstract: In the past 20 years, human amniotic membrane (hAM) has become widely recommended as an ophthalmic surgical patch, and as a substrate for stem cell tissue equivalents for ocular surface reconstruction. HAM reduces ocular surface scarring and inflammation, and enhances epithelialization. In addition, it shows limited immunogenicity and some anti-microbial properties. Thanks to these properties, hAM has been also used in wound healing, especially for burns and ulcers. Since its first clinical applications, hAM has been used for other indications, such as oral and maxillofacial, earnose-throat, gynaecological and orthopaedic surgeries. This review will describe: (i) past and current clinical uses of hAM; (ii) accepted processing methods, including preparation, preservation, sterilization and de-epithelialization and their impact on the properties of hAM, especially growth factor release, cell viability and immunological properties; (iii) its applications in tissue engineering, namely as scaffold or carrier of biological molecules. Economical aspects are presented at the end of this review. Existing patents are reported for each section and existing marketed products are listed. Patents, products and ongoing clinical trials, were identified by electronic searches on the internet in January 2013. In conclusion, in view of published data, hAM seems to have real market potential in regenerative medicine, in particular in emerging fields such as oral and maxillofacial, ear-nose-throat, gynaecological and orthopaedic surgery.

Keywords: Clinical uses, economical aspects, growth factors, human amniotic membrane (hAM), marketed products, patents, processing; tissue engineering. INTRODUCTION The foetal membranes (i.e. amnion and chorion Fig. (1a) extend from the placental disc to cover the foetus in the amniotic cavity, ensuring physical and biological protection during foetal development. The amnion, also widely called the “amniotic membrane (AM)” Fig. (1b), is about 20 to 50 m thick, and corresponds to the inner layer of the foetal membrane. It consists of a thin epithelium with a basement membrane and a stroma of vascular connective tissue called the amniotic mesoderm. This epithelium contains a simple, continuous, uninterrupted line of columnar, cuboid or flat cells that arise from the embryonic epiblast and are in contact with the amniotic fluid. This epithelium sits over a resistant and well-defined basement lamina connected to the amniotic mesoderm where three structures can be identified: (i) an acellular compact layer composed of collagen type I and III and fibronectin; (ii) a netting of mesenchymal stromal cells (MSC) (derived from the extraembryonic mesoderm), *Address correspondence to this author at the Orthopaedic and Traumatology Surgery Service, University Hospital of Besancon, Bd Alexandre Fleming, 25030 Besancon Cedex, France; Tel: (+33) 3 81 21 89 98; Fax: (+ 33) 3 81 66 93 06; E-mail: [email protected] 2210-2965/13 $100.00+.00

called the fibroblastic layer; and (iii) an intermediate layer or sponge zone, rich in proteoglycans, glycoproteins and non fibrillar collagen type III, which is loosely connected to the chorion [1]. The isolation protocols, phenotypic markers, and in vitro differentiation potential of epithelial and mesodermal cells from the human AM (hAM) have been widely described [24]. Both the phenotype and the potential to differentiate towards the three mesodermal lineages (adipogenic, osteogenic and chondrogenic) of hAM-derived cells have been shown to be similar to bone marrow-derived MSC [5-7]. Other differentiation potentials have also been described for these amniotic-derived cells [8, 9]. The hAM has many beneficial biological properties, including anti-bacterial, anti-viral, anti-inflammatory, analgesic and anti-fibrotic properties, and it allows wound healing, re-epithelialization and reduction of scarring. In addition, Mamede et al. reviewed pluripotency of amnion-derived cells, describing anti-angiogenic and pro-apoptotic and nontumorigenic properties [10]. hAM has also been reported to be a translucent and elastic scaffold that acts as a barrier for maintaining humidity © 2013 Bentham Science Publishers

2 Recent Patents on Regenerative Medicine 2013, Vol. 3, No. 3

Gindraux et al.

A

B

Fig (1). (A) Foetal membrane (amnion and chorion) and (B) Amnion (or amniotic membrane) on nitrocellulose support (4.7cm of diameter) just after separation from the chorion (Own data).

with high gas permeability, which provides a matrix for cellular migration and proliferation, and contains a number of essential growth factors and cytokines [1, 2, 11-15]. In addition, hAM has the advantage of eliciting few ethical issues, with unlimited availability, easy procurement from the placenta, low processing costs and low immunogenicity. These properties have played a significant role in advancing the use of hAM in surgery, initially for reconstruction of the corneal and conjunctival surfaces, treatment of open ulcers and traumatic wounds, and skin transplantation [8, 10, 16]. hAM has since been proposed for use in other types of surgery in ophthalmology to treat corneal, conjunctival and limbal lesions, burns, scars and defects as well as in general surgery, to reconstruct skin, genito-urinary tract and other surfaces, or soft tissues [16-18].

Sabella in 1913, for the management of skin burns and ulcerated skin surfaces [10]. The amnion side of foetal membranes (amnion and chorion) was opposed onto the skin and covered with war paraffin. After 48 h, the chorion, bonded to the paraffin, was removed, and the amnion adhered to the wound. The authors observed a lack of infection, significant pain relief and an increased rate of re-epithelialization of the damaged skin surface. Thereafter, there were no further publications for the next 22 years. Starting in 1935, several authors published clinical applications of hAM (see [19] for review) and successful clinical trials were performed for: -

Vaginoplasty

-

Conjunctival reconstruction

After a brief presentation of past and current clinical uses of hAM, we detail processes for its preparation and preservation, tissue engineering applications, existing marketed products and economical aspects. Patented research is reported for each section. We focus here on patents and regenerative medicine or repair surgeries with successful clinical trials performed with hAM in its entirety, and not on MSC derived from the tissue.

-

Treatment of burns and traumatic skin wounds

-

Intra-abdominal treatment of adhesion.

The patent search was carried out in January 2013 using the following terms: “amniotic AND membrane”, or “amnion”. The databases searched were: the European Patent Office (EPO, http://www.epo.org), the United States Patent and Trademark Office (USPTO, http://www.uspto.gov) and Google Patents (www.google.com/patents). Using the same terms and at the same date, we performed a search for clinical trials on the U.S. National Institutes of Health Clinical Trials Database (http://www.clinicaltrials.gov), the World Health Organization International Clinical Trials Registry Platform (ICTRP, http://apps.who.int/trialsearch/) and the European Union Clinical Trials Register website (https://www.clinicaltrialsregister.eu). Finally, marketed products were identified by a search on the internet with Google Search, also undertaken in January 2013.

In 1985, Lu [20] patented a suture of hAM and its manufacturing process: the suture is made of hAM lamina by chrome tanning process after having removed the fat and protein components on its surface. It is absorbed naturally and does not have to be removed after suturing. It has no immunity repellent effect, no pain after surgical operation and no evident scarring, and thus, is suitable for use in various surgical operations, especially surgery of face, plastic surgery, ophthalmology and suturing of various viscera.

I. CLINICAL USES OF THE HUMAN AMNIOTIC MEMBRANE 1. Medical History Mamede et al. reported that hAM was first used by Davis in 1910 for skin transplantation, followed by Stern and

Trelford et al. reported that in 1952, Douglas used hAM for large wounds and, for the first time, the mesenchymal layer of the separated amnion was recognized as providing a more consistent and effective “take”. Trelford et al. confirmed these findings 20 years later [19].

From 1972 onwards, and especially since its rediscovery in 1995 [21], other authors have confirmed all the clinical applications previously presented, and also reported new indications such as the genito-urinary tract, stomach, larynx, oral cavity, head and neck, in clinical trials or case reports. hAM was used as temporal dressing or allografts [10, 19, 22]. Table 1 summarizes the largest, most recent, best known and most suitable applications of hAM in oral and maxillofacial, ear-nose-throat, gynaecological and orthopaedic surgeries [18, 23-37]. (Ocular surface reconstruction and wound healing are treated separately in the next paragraph).

Clinical Uses of Human Amniotic Membrane

Table 1.

Recent Patents on Regenerative Medicine 2013, Vol. 3, No. 3

Most Recent and Best Known Uses of Ham for Oral and Maxillofacial, Ear-Nose-Throat, Gynaecological and Orthopaedic Surgeries.

Authors

Year

Treatments

Indications

Tests

Number of patients

References

Arai et al.

2012

Hyperdry and g-sterilized

Defects in the tongue and buccal mucosa

Alone

10

[25]

Tsuno et al.

2012

Hyperdry and g-sterilized

Vestibulopasty

Alone

2

[36]

Kanazawa et al.

2012

Hyperdry and g-sterilized

Tympanoplasty

hAM alone or with fibrin glue versus temporal fascia

27

[29]

Amer et al.

2012

Fresh (sterile saline with antibiotics, 48-72 h, 4 °C)

Severe intrauterine adhesions

Fresh versus lyophilized versus intrauterine balloon

1

[24]

Kothari et al.

2011

Glycerol, 4 °C

Buccal mucosal defect

Alone

1

[31]

Sharma et al.

2011

Glycerol, 24 h

Vestibulopasty

Alone

10

[35]

Kothari et al.

2011

Glycerol, 4 °C

Vestibulopasty

Alone

10

[31]

Shojaku et al.

2011

Hyperdry and g-sterilized

Tympanoplasty

hAM versus temporal fascia

20

[18]

Velez et al.

2010

Cryopreserved (method developed by Tseng et al. [168])

Periodontal soft tissue healing

Alone

15

[37]

Amer et al.

2010

Fresh (sterile saline with antibiotics, 48-72 h, 4 °C) or lyophilized and g-sterilized

Severe intrauterine adhesions

Fresh versus lyophilized versus intrauterine balloon

45

[23]

Kothiwale et al.

2009

Lyophilized and g-sterilized

Periodontal soft tissue healing

Associated with alone bone allograft or xenograft

10

[32]

Gurinsky et al.

2009

Deshydrated BioXclude™

Periodontal soft tissue healing

Alone

5

[27]

Jay et al.

2009

Sterilized AmnioClear®

Posterior tibial and Achilles tendons tears

Alone

2

[28]

Samandariet al.

2004

Fresh (sterile saline with antibiotics, 24 h, 4 °C)

Vestibulopasty

Alone

7

[34]

Bleggi-Torres et al.

1997

Fresh (sterile saline with antibiotics, 48-72 h, 4 °C)

Severe intrauterine adhesions

Alone

10

[26]

Some patents on clinical indications of hAM have been filed by: -

-

3

Young [38], in 1995, on hAM graft or wrap to prevent adhesions or bleeding of internal organs: The hAM is sterilized (and may be cross-linked) and is applied as a single layer to an injured surface subject to adhesions or wrapped on internal organs subject to bleeding, and can be sutured or applied by a liquid anti-adhesive adjuvant. The applied surface can be wet as well as dry. Lapierre [39], in 2005, as a healing dressing, e.g. for mucosa burn, which has an inert component and serum covering hAM to be applied on wound, where component and serum have healing properties by presence of growth factors and intrinsic characteristics.

2. Fluently Clinical Uses The indication that is most frequently reported concerns ocular surface reconstruction. Since 1995, hAM transplantation has been successfully applied to patients with a variety of ocular surface diseases, including the following [10, 17, 40-45]:

-

Corneal surface reconstruction: persistent epithelial defect with corneal ulceration, acute chemical burns, removal of epithelial or subepithelial lesions (band keratopathy, scars, tumors), painful bullous keratopathy and partial or complete limbal stem cell deficiency (with stem cell grafting)

-

Conjunctival surface reconstruction: acute chemical burns and acute Stevens-Johnson syndrome, covering defects after removal of large conjunctival lesions (tumors, conjunctival intraepithelial neoplasia, scars, conjunctival folds parallel to the edges of the eyelids), symblepharon, fornix reconstruction, anophthalmia, bleb revisions, scleral thinning, pterygium.

There are three types of applications for hAM transplantation in ocular surface disorders [10, 40]. Mamede et al. have proposed the following classification [10]: -

As a graft, where the transplantation of hAM on corneal or scleral stroma induces proliferation and differentiation of the ocular surface epithelium,

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-

As a patch, where hAM can be used to cover the inflamed ocular surface, especially when associated with epithelial defects, acute phase of chemical/thermal burns or Stevens Johnson syndrome,

-

As a stuff, for deep corneal/scleral ulcerations or small perforations refractory to conventional medical therapy and treated by stuffing small pieces of hAM into the stromal defects, followed by hAM grafting and patching over the area.

Firstly, when hAM is applied as a permanent graft, it is used to fill the tissue defect induced by the disease process or by surgery. Consequently, the host cells grow over or into the hAM, and the membrane is integrated into the host tissue. Secondly, when hAM is used as a temporary biological bandage/patch, the main goal is to suppress acute or chronic host tissue inflammation caused by disease or surgery, in order to promote healing with minimal scarring [40]. In 2004, Tseng et al. patented a biopolymer covering for a tissue surface including, for example, a dressing, a bandage, a drape such as a bandage contact lens, a composition or covering to protect tissue, a covering to prevent adhesions, to exclude bacteria, to inhibit bacterial activity, or to promote healing or growth of tissue [46]. An example of such a composition is a hAM covering for an ocular surface. Use of a covering for a tissue surface according to the invention eliminates the need for suturing. The invention also included devices facilitating the fastening of a membrane to a support, culture inserts, compositions, methods, and kits for making and using coverings for a tissue surface and culture inserts. Compositions according to the invention may include cells grown on a membrane or attached to a membrane, and such compositions may be used as scaffolds for tissue engineering or tissue grafts. The second most commonly reported indication is for wound healing. As shown above, the use of hAM in dermatology as a dressing for wounds and burns dates back to the early 20th century. With improvements in sterilization techniques in the late 20th century, the possibility of using hAM to treat vascular ulcers came to be considered. Some case reports described the use of hAM as a novel treatment for chronic wounds in patients with epidermolysis bullosa and for epithelial loss in Stevens–Johnson syndrome [47]. The hAM can provide excellent coverage of burn wounds in preparation for skin grafting or as a biological dressing when there is a limited supply of skin grafts [48]. Successful data of hAM use have been reported in patients with extensive burns (superficial or partial-thickness), where it has been shown to be safe, easy to use, and extremely beneficial in allowing fast re-epithelialization of denuded skin [49-55]. The treatment of full-thickness burns was associated with a lower success rate, since hAM underwent autolysis and disintegration [56]. Less is known about the effectiveness of hAM in the treatment of chronic ulcers [57, 58]. In 2007, Mermet et al. performed the first prospective study of long-standing vascular ulcers refractory to standard treatment, demonstrating that hAM significantly reduced ulcer size, pain intensity, and reepithelialization time through its capacity to promote growth,

Gindraux et al.

adhesion, and differentiation of epithelial cells and to prevent their apoptosis [59]. Subsequently, Pesteil et al. confirmed these results [60, 61]. The benefits obtained by these authors, and the benefits reported in the literature in other dermatological conditions led Alsina-Gibert et al. to propose the use of hAM to treat vascular ulcers refractory to standard treatment [62]. 3. Present and Future Currently, the only clinical indication for hAM grafting is in ophthalmology. Large clinical series in patients with corneal and conjunctival disorders have recently been published [63, 64] and phase I to III clinical trials are currently under way and are reported in Table 2. Regarding the use of hAM in dermatology, some clinical trials, summarize in Table 2, are also ongoing in new indications, such as pressure ulcers of diabetic foot ulcers. According to the literature on the subject and clinical trials already performed in dermatology, we purport that hAM grafting will soon be considered as a re-emerging therapeutic option in this field, and approved as a new clinical indication by the competent health authorities. Finally, some clinical trials performed in oral, maxillofacial and orthopaedic surgery for gingival recession and tendon tear have already been completed and published, see Table 1, while some others are ongoing, see Table 2, with a novelty concerning the treatment of plantar fasciitis. These new clinical indications represent the future of the hAM allograft. To date, the immune properties of hAM allow it to be used in as an allogenic graft. Another future application of this foetal tissue would be as an autograft, as has previously been described in paediatric investigations for the closure of a myelomeningocele, for example [65, 66]. Regardless of whether hAM is used as an autograft or allograft, certain safety procedures for its preparation and immediate implantation, as well as for storage and subsequent implantation must be strictly observed [40, 67, 68]. These techniques are presented in the next section. II. PREPARATION AND PRESERVATION OF HUMAN AMNIOTIC MEMBRANE To ensure the highest quality of tissue, hAM obtained after caesarean section under sterile conditions is exclusively recommended by current protocols [45, 68]. The pregnant donors must be tested prior to donation for human immunodeficiency virus (HIV), hepatitis B and C, syphilis, cytomegalovirus and Toxoplasma gondii [69]. Although fresh hAM is frequently applied [19], see Table 1, the potential risk of disease transmission should be taken into consideration. To overcome doubts about infectiousness and to respect the transplantation laws of many countries, several preserving techniques have been developed over the years. Long-time storage avoids the possibility that the donor is in the “window period” of infection. Hence, even if serological tests are apparently normal, it is advisable to repeat testing after 6 months. hAM can be stored until both the initial and the 6month samples are negative.

Clinical Uses of Human Amniotic Membrane

Table 2.

Recent Patents on Regenerative Medicine 2013, Vol. 3, No. 3

5

Update on Treatment, Screening or Basic Science Clinical Trials Using Ham for Ophthalmology, Dermatology, Oral And Maxillofacial and Orthopaedic Surgeries. (* Screening, ** Basic Science). nd: not done

Year

Title

Pathology

Sponsor, Collaborator, Country Recruitment Enrolment

Source ID

Ophthalmology Recurrent pterygium surgery with mitomy2011 cin c application using limbal conjunctival versus amniotic membrane

Recurrent pterygium

Sun Yat-sen University China

Recruiting

96

An evaluation of the effect of the amniofix amniotic membrane allograft in patients 2011 undergoing posterior instrumentation removal

Adhesion of soft tissues

MiMedx Group, Inc. USA

Recruiting

50

2010

Amniotic membrane extract eye drops for eye surface diseases

Ocular surface disease

2010

The surgical repair of refractory ocular surface diseases using amniotic membrane

Refractory ocular surface disease

University of Toyama Japan

Recruiting

250

Refractory glaucoma

Shaheed Beheshti Medical University, Iran

Recruiting

nd

Not recruiting

40

Ahmed glaucoma valve alone, with amniotic 2009 membrane or with Mitomycin C (MMC) for treatment of refractory glaucoma Sutureless cryopreserved amniotic mem2009 brane graft (Prokera®) and wound healing after photorefractive keratectomy

Zhongshan Ophthalmic Center Not recruiting China

Walter Reed Army Medical Center, R&D Tissue Tech, Corneal Department of Ophthalmology wound healing and Optometry St John's Hospital and Clinics, USA

50

Amniotic membrane transplantation versus 2009 conjunctival–limbal autograft for primary pterygium

Primary pterygium

Hermanos Ameijeiras General Hospital Cuba

Completed

nd

A clinical trial to study the role of amniotic 2009 membrane in patients with chemical or thermal injury to the eye

Acute ocular burn

Centre for Ophthalmic Sciences India

Completed

100

Amniotic membrane and anterior stromal 2008 puncture to the treatment of symptomatic bullous keratopathy

Bullous keratopathy

Federal University of Sao Paulo, Fundaao de Amparo Pesquisa do Estado de Sao Paulo, Brazil

Completed

38

Alcohol 20 % for separation of pterygium 2008 and comparison of different wound closure methods

Pterygium

Soroka University Medical Center Israël

Recruiting

150

Autologous transplantation of cultivated 2008 limbal stem cells on amniotic membrane in Limbal Stem cell Deficiency (LSD) patients

Limbal stem cell deficiency

Royan Institute, Labafi Nejad Eye Research Center Iran

Completed

10

Symblepharon

Federal University of Sao Paulo Brazil

Not recruiting

10

Scleral thinning

Federal University of Sao Paulo Brazil

Enrolling by invitation

49

Transplant of epithelium conjunctival human autologous cultivated ex vivo in 2008 amniotic membrane for the treatment of symblepharon Comparison amongst scleral, corneal and 2008 amniotic membrane grafts to restore scleral thinning

clinicaltrials.gov NCT01319721

clinicaltrials.gov NCT01357187 anzctr.org.au ACTRN12610000014055 umin.ac.jp JPRN-UMIN000002983 clinicaltrials.gov NCT00893490

clinicaltrials.gov NCT00915759

registroclinico.sld.cu RPCEC00000081 ctri.nic.in CTRI/2009/091/001018

clinicaltrials.gov NCT00659308

clinicaltrials.gov NCT00704977 clinicaltrials.gov NCT00736307

clinicaltrials.gov NCT00799526

clinicaltrials.gov NCT00801073

6 Recent Patents on Regenerative Medicine 2013, Vol. 3, No. 3

Gindraux et al.

Table (1). Contd…..

Year

Pathology

Sponsor, Collaborator, Country Recruitment Enrolment

Amniotic membrane associated with 2008 conjunctival autograft versus conjunctival autograft for recurrent pterygia

Recurrent pterygia

Federal University of Sao Paulo Brazil

Enrolling by invitation

40

To compare fibrin glue and suture in 2007 primary pterygium excision with amniotic membrane transplantation

Pterygium

Chulalongkorn University Thailand

Completed

32

The application of oral mucosal epithelial cell sheets cultivated on amino membrane in 2007 patients suffering from corneal stem cell insufficiency or symblepharon.

Limbal insufficiency, symblepharon

National Taiwan University Hospital Taiwan

Terminated

0

Transplantation of tissue cultured human 2006 amniotic epithelial cells onto damaged ocular surfaces

Corneal epithelial dystrophy

University of Texas Southwestern Medical Center, USA

Recruiting

20

Transplantation of cultivated limbal epithe2006 lium on amniotic membrane for limbal stem cell deficiency

Ocular surface disease

Singapore National Eye Centre, Singapore Eye Research Institute Republic of Singapore

Suspended

8

Eye burn

Shaheed Beheshti Medical University, Iran

Recruiting

90

Pterygium

Baskent University Turkey

Completed

30

Clinical study of micronized amniotic mem2012 brane transplantation in the treatment of chronic and refractory wounds

Chronic and refractory wound

Department of Burn, Shanghai Hospital China

Recruiting

60

Human amniotic membrane grafting and 2012 standard of care versus standard of care alone in the treatment of venous leg ulcers

Venous leg ulcer

MiMedx Group, Inc. USA

Recruiting

100

Diabetic foot ulcer

MiMedx Group, Inc. USA

Completed

25

Burn

Ghotbeddin Hospital, Burn Center, Shiraz Iran

Completed

54

Extensive wound

Fundación para la Formación e Investigación Sanitarias, Murcia Spain

Authorised

nd

Comparative study of two application regi2012 mens of amniotic membrane wound graft in the management of diabetic foot ulcers

Diabetic foot ulcer

MiMedx Group, Inc. USA

Recruiting

40

Trial of amniotic membrane wound graft in the management of diabetic foot ulcers

Diabetic foot ulcer

MiMedx Group, Inc. USA

Recruiting

120

Bed sore

Christian Medical College, Vellore India

Not recruiting

30

2006

Title

The role of amniotic membrane transplantation in ocular chemical burns

ICG angiography in amniotic membrane graft 2006 and conjunctival autograft after pterygium excision*

Source ID clinicaltrials.gov NCT00802620 clinicaltrials.gov NCT00457223

clinicaltrials.gov NCT00491959

clinicaltrials.gov NCT00344708 clinicaltrials.gov NCT00348114 clinicaltrials.gov NCT00370812 clinicaltrials.gov NCT00383825

Dermatology

Comparative study of amniotic membrane 2012 wound graft in the management of diabetic foot ulcers Evaluation of effect of amniotic membrane 2012 on split-thickness skin fixation in patient with extremities burns in Ghotbeddin hospital

2012

2012

Clinical trial on the use of the amniotic membrane for large wound epithelization

Use of amniontic membrane as biological 2011 dressing for the treatment of bed sores in comparison with conventional dressings

chictr.org ChiCTR-TRC12001889 clinicaltrials.gov NCT01552447 clinicaltrials.gov NCT01552499 irct.ir IRCT138902281605N9 clinicaltrialsregister.eu EUCTR2011-00439511-ES clinicaltrials.gov NCT01657474 clinicaltrials.gov NCT01693133 ctri.nic.in CTRI/2011/11/002167

Clinical Uses of Human Amniotic Membrane

Recent Patents on Regenerative Medicine 2013, Vol. 3, No. 3

7

Table (1). Contd…..

Year

Title

Pathology

Sponsor, Collaborator, Country Recruitment Enrolment

Comparison of itching and scar in simple skin graft and skin graft with amnion mem2010 brane in burn patients who admitted in Ghotbeddin burn hospital in one year

Burn and hypertrophic scar

Ghotbeddin Hospital, Burn Center, Shiraz Iran

Completed

30

Comparative result of graft take in 2 different methods for burn patient with old and in2010 fected granulation tissue in Ghotbeddin hospital at 2009 and 2010

Burn

Ghotbeddin Hospital, Burn Center, Shiraz Iran

Completed

90

Evaluation of the cryopreserved amniotic 2009 membranes in the care of resistant vascular ulcers

Resistant vascular ulcer

University Hospital, Limoges, Etablissement Français du Sang France

Recruiting

25

ACCS (Amnion-derived Cellular Cytokine 2009 Solution) versus standard care in treating partial thickness burns

Burn

Stemnion, Inc. USA

Completed

17

Study of donated amnion, foetal placental 2007 membrane, as skin substitute for burn patients**

Wound

The University of Texas, Galveston USA

Not recruiting

1500

2007 Use of amnion on partial thickness burns

Burn

The University of Texas, Galveston USA

Withdrawn

0

Plantar fasciitis

MiMedx Group, Inc. USA

Recruiting

45

Tendon tear

Orthopedic Foot and Ankle Center, Ohio, USA

Not recruiting

40

Gingival recession

Faculty of Dentistry, Tabriz Iran

Recruitment complete

30

Source ID

irct.ir IRCT138811131605N7

irct.ir IRCT138808301605N6

clinicaltrials.gov NCT00820274 clinicaltrials.gov NCT00886470 clinicaltrials.gov NCT00592189 clinicaltrials.gov NCT00674999

Other: Oral and Orthopaedic Surgeries Comparative study of amniotic membrane 2012 injectable in the treatment of recalcitrant plantar fasciitis The role of cryopreserved human amniotic 2012 membrane in the surgical repair of peroneal tendons: an infrared thermography model 2010

Amniotic membrane in treatment of gingival recessions

Guidance and operating standards for the procurement, processing and distribution of tissue such as hAM have been issued by the US Food and Drug Administration (FDA) [70], and reviewed by Dua [71]. When appropriately processed and preserved, hAM can be used for a number of indications, either as a graft to replace damaged ocular surface stromal matrix or as a patch (dressing) to prevent unwanted inflammatory insults from gaining access to the damaged ocular surface, or a combination of both. 1. Current Techniques for Preparation and Preservation The most common procurement for hAM banking is cryopreservation with glycerol and Dulbecco’s Modified Eagle Medium (DMEM). These procedures and the most common alternatives such as cryopreservation in Dimethyl sulfoxide (DMSO) or lyophilization are described in the following sections. In order to minimize the risk of infections that may be transmitted by hAM, recent studies have used the combination of tissue preservation with sterilization by gamma (g)-irradiation. Novel agents, such as peracetic acid (PAA) and trehalose have also been used in preserving and

clinicaltrials.gov NCT01659827 clinicaltrials.gov NCT0170818 irct.ir IRCT138808142670N1

sterilizing hAM. Other techniques have been reported in the literature but are not detailed in the present review, including heat-dried hAM, air-dried hAM and preservation in cold glycerol (+4 °C) (see [72] for references). Some of the techniques described here have been the object of patents, Table 3, or have been used to produce hAM for commercialization, Table 4. Cryopreservation in Glycerol After having reintroduced hAM for ocular surface reconstruction in the 1990s, Lee and Tseng suggested the cryopreservation of the foetal membranes. Cryopreservation would enable the hAM to retain its properties and would render the amniotic epithelial cells nonviable and thus non-immunogenic [73]. Initially, the placenta is cleansed of blood clots with an antimicrobial solution before hAM is separated from the chorion. Subsequently, hAM is apposed onto nitrocellulose paper with the epithelial side up. The sheets are placed in sterile vials containing DMEM and glycerol (86 %) at a ratio of 1:1. Although glycerol is known to have anti-viral effects, it is

8 Recent Patents on Regenerative Medicine 2013, Vol. 3, No. 3

Table 3.

Gindraux et al.

Patents on hAM: Indications, Preparation, Preservation and De-epithelialization Processes and Tissue Applications.

Field of Action

Clinical indication

Processing

Preservation

Decellularization

Tissue engineering

Year

Title

Patent Application Number

Reference

V. Lapierre

2005

Healing dressing for e.g. mucosa burn, has inert component and serum covering amniotic membrane to be applied on wound, where component and serum have healing properties by presence of growth factors and intrinsic characteristics

FR2892311

[39]

S. Tseng et al.

2004

Amniotic membrane covering for a tissue surface and devices facilitating fastening of membranes

US20040181240

[46]

R. Young

1995

Amniotic membrane graft or wrap to prevent adhesions or bleeding of internal organs

AU1343195

[38]

T. Samson et al.

2012

Methods of manufacture of immunocompatible amniotic membrane products

EP2536417

[120]

M. Ramos et al.

2004

Method for the preparation of immunologically inert amniotic membranes

US20040126878

[119]

Y. Tong

2012

The utility model claims a composite anti- freezing fluid and application thereof and using the resisting freezing liquid storing the amniotic membrane method for composite anti-freezing liquid, and method for preserving human amnion by using same

CN102763639

[108]

H. Zhou

2010

Amniotic membrane long-term preserving fluid and preparation method thereof

CN102132697

[107]

G. H. Kim et al.

2004

Cryopreservation medium composition of amniotic membrane for healing ocular surface disease

KR20040020413

[80]

E. S. Miljudin et al.

2002

Method for preserving amniotic membrane

RU2214091

[79]

S. Tseng

2001

Grafts made from amniotic membrane; methods of separating, preserving and using such grafts in surgeries

US6326019

[74]

J. Han et al.

2012

Method for preparing acellular amniotic membrane

CN102327640

[131]

R. Cong et al.

2010

Method for preparing carrier bracket of tissue engineering artificial corneal endothelium by using fresh amniotic membrane

CN101843923

[130]

S. Guhathakurta et al.

2009

A treated amniotic membrane and method of treating amniotic membrane

WO2009044408

[129]

J. C. Kim et al.

2009

Novel preparation method for a decellularized amniotic membrane using freeze-thaw-centrifuge principle and use thereof

KR20110014024

[128]

Y. Xue

2002

Engineered scaffold of amniotic membrane tissue and process for removing cells from amniotic membrane

CN1369555

[127]

S. Tseng et al.

2012

Amniotic membrane preparations and purified compositions and antiinflammation methods

US20120328690

[164]

S. Tseng et al.

2012

Amniotic membrane preparations and purified compositions and antiangiogenesis treatment

US8187639

[163]

S. Tseng et al.

2012

Amniotic membrane preparations and purified compositions and therapy for scar reversal and inhibition

US8182840

[166]

S. Tseng et al.

2012

Purified amniotic membrane compositions and methods of use

US8153162

[165]

Z. Xia et al.

2012

Amniotic membrane microcarrier capable of simulating niche microenvironment for growth of epidermal stem cells and skin substitute thereof

CN102367434

[142]

J. Eibl et al.

2011

Process for differentiating stem cells of the amniotic membrane

WO2011151043

[151]

M. E. Kreft et al.

2011

Process for establishing of differentiated urothelium on connective tissue of intact human amniotic membrane

SI20110000110

[159]

Inventors

Clinical Uses of Human Amniotic Membrane

Recent Patents on Regenerative Medicine 2013, Vol. 3, No. 3

9

Table (3) contd……

Field of Action

Table 4.

Patent Application Number

Reference

Surgical Membrane

US2008193554

[162]

2006

Retinal pigment epithelial cell cultures on amniotic membrane and transplantation

US2006002900

[138]

C. H. Fang

2006

Pharmaceutical amniotic membrane and processes for their preparation

CN101040616

[161]

G. Peyman

2005

Use of amniotic membrane as biocompatible devices

WO2006002128

[122]

F. Zhang

2004

Amniotic membrane mediated delivery of bioactive molecules

WO2004000164

[160]

R. F. Tsai et al.

2001

Method for expansion of epithelial stem cells on an amniotic membrane and resulting graft

WO0180760

[137]

D. Lu

1985

Suture of amniotic membrane and its manufacturing process

CN85108766

[20]

Inventors

Year

H. Dua et al.

2008

S. Binder et al.

Title

Currently Commercially Available hAM Products.

Company

Product

Website

Product information

Uses

AFCellTM

AmnioClear®

http://www.afcellmedical.com/ products.html

Aseptically processed and sterilized human amniotic membrane

Soft tissue surgery

Alphatec Spine®

AmnioShield®

http://www.alphatecspine.com/ products/biologics/amnioshield.asp

PurionSM processed human amniotic membrane

Wound management, soft tissue surgery

http://www.amnioxmedical.com/ NEOX-regenerative-matrix.html

Cryopreserved human amniotic membrane (CryoTek™ processing method)

Ophthalmic surgery, wound management, soft tissue surgery (orthopaedic, spinal surgery)

BioDfence

http://www.biodlogics.com/ biodfence.htm

Dehydrated, sterilized human amniotic membrane

BioDfactor

http://www.biodlogics.com/ biodfactor.htm

Injectable micronized, sterilized and dehydrated human amniotic membrane

Amniograft®

http://www.biotissue.com/ products/amniograft.aspx

Cryopreserved human amniotic membrane on a carrier paper

Amnioguard™

http://www.biotissue.com/products/ AmnioGuard.aspx

Cryopreserved human amniotic membrane to cover glaucoma drainage devices

Prokera®

http://www.biotissue.com/products/ prokera.aspx

Cryopreserved human amniotic membrane (CryoTek™ processing method) in a convenient, sutureless, thermoplastic ring set of 16mm diameter (FDA Class II medical device)

Acelagraft

http://www.celgene.com/

Decellularized and dehydrated human amniotic membrane

Ophthalmic surgery

Ambiodry2®

http://www.iopinc.com/store/ambiodry2/

®

http://www.iopinc.com/store/ambiodisk/

Cleaned, dehydrated, cell-devitalized and sterilized human amniotic membrane with "watermark" impression for visual identification of the basement and stromal surfaces

Ophthalmic surgery

Lyophilized human amniotic membrane

Ophthalmic surgery

NEOX®100 Amniox™ Medical

®

NEOX 1k

BioDlogics

Biotissue™

Celgene

IOP Ophthalmics

Keera Srl

AmbioDisk Ambio5®

http://www.iopinc.com/store/ambio5/

AMX: Amniotic Membrane eXtract

http://www.emilianoghinelli.com/ Amniotic-Membrane-eXtract.html

Wound management, soft tissue surgery

Ophthalmic surgery

10 Recent Patents on Regenerative Medicine 2013, Vol. 3, No. 3

Gindraux et al.

Table (4) contd…… Company

Product

Website

EpiFix ® AmnioFix

MiMedx Group

Product information

http://www.mimedx.com/epifix/overview/ ®

Wound management Dehydrated human amniotic membrane PurionSM processed

AmnioFix® Wrap http://www.mimedx.com/ amniofix/surgical-application/ ®

AmnioFix Injectable

Uses

Soft tissue surgery (orthopaedic, spinal, general, urologic and gynaecologic surgery)

Injectable micronized and dehydrated human amniotic membrane PurionSM processed

Soft tissue inflammation (tendonitis, bursitis, fasciitis), management of chronic ulcer, wound, and burn

NHS Blood and Transplant

Amniotic membrane small or large

http://www.nhsbt.nhs.uk/ tissueservices/products/eyes/ amnioticmembrane/

Decontaminated and frozen (50 % glycerol/Hanks solution) human amniotic membrane stored on nitrocellulose paper

Ophthalmic surgery

SKY Orthobiologics

Skye Reset™

http://skyeorthobiologics.com/products/ specialty/reset_specialty.html

Sterilized human amniotic membrane

Wound management, soft tissue surgery

BioXclude™

http://www.snoasismedical.com/ bioxclude.php

Dehydrated and sterilized graft composed of allograft amnion and chorion tissue (300 m thick) PurionSM processed

BioCover™

http://www.snoasismedical.com/

Dehydrated human amniotic membrane (no need to rehydrate, sutureless)

Snoasis Medical

not reputed as a sterilizing agent. For storage, these vials were frozen at -80 °C [73]. In 2001, Tseng patented the method of separating, preserving and using such grafts made from hAM [74]. The hAM is separated from the chorion and prepared as described in the previous paragraph. The cells of the hAM are killed, preferably while being frozen and thawed in the storage solution comprising a culture medium (e.g. DMEM) and a hyperosmotic agent (e.g. glycerol), in which the hydration of the hAM is maintained. The membrane can be impregnated with therapeutic agents, prior to storage, for use in post-surgical healing or other therapies. All reports published so far have used cryopreserved membranes, and the vast majority of the experience gained in Europe and the USA is based on this technique. Indeed, the FDA has recommended it as a reliable preservation method [43, 75], and it has been used for the evaluation of hAM processing techniques [76]. This long-term storage cryopreservation method has shown effectiveness in both the ocular and dermatological fields [56]. Concerning glycerol cryopreservation, we showed (Laurent et al. submitted) in histological studies Fig. (2) that cryopreserved hAM Fig. (2g, 2h, 2i) presented both cell and matrix alterations in comparison with fresh hAM Fig. (2a, 2b, 2c). As for epithelial cells, MSC (*) in the amnion seemed to be damaged in cryopreserved hAM. Specific labeling of the extracellular matrix components also exhibited severe damage of the amnion matrix ( ) in cryopreserved hAM: type I collagen fibers were disrupted and collapsed Fig. (2j), as were the elastic fibers, which were frayed and disorganized Fig. (2l) compared to fresh hAM Fig. (2d, 2f). The thick basement hAM could still be highlighted by type IV collagen labeling Fig. (2k), but microfibrils seen in fresh

Oral surgery (soft tissue and bone management), wound management

hAM Fig. (2e) were no longer present in the cryopreserved hAM. These first results showing significant extracellular matrix damage suggest that cryopreserved hAM can be used as a biologic support rather than as a functional tissue. Cryopreservation in DMSO DMSO has been used as an alternative to hAM preservation in glycerol DMEM. Instead of washing the hAM with an antibiotic-saline solution after placenta retrieval, an increasing concentration of DMSO is used. Azuara-Blanco et al. [77] used 4 %, 8 %, and then 10 % DMSO, while Kubo et al. [78] used 0.5, 0.1 and 0.15 M DMSO for washing. The hAM can then be stored in 10 % (0.15 M) DMSO at -80 °C for several months. Clinically, there have been a number of reports on the successful use of DMSO-cryopreserved hAM in ocular surface reconstruction (see [44] for references). The effects of cryopreservation on the structural integrity and biological contents of hAM have not been studied extensively. However, extrapolating from results of prior clinical results, it can be assumed to be of similar quality to glycerol-cryopreserved hAM. Cryopreservation Process Improvement In 2002, Mijudin et al. [79] patented a method that consists in immersing cleaned pieces of hAM remnants from the chorion first into 0.2 % chlorhexidine solution, then into a solution containing 40 mg/mL of gentamicin and 2.5 mg/mL amphotericin B and lastly into glycerol for 20 min. The pieces of hAM are then fixed on metal frames and dried under sterile conditions over silicagel for 18-24 h, then separated from the frames and packed. This technique enhanced the effectiveness and duration of preservation without using fixation on nitrocellulose paper.

Clinical Uses of Human Amniotic Membrane

Recent Patents on Regenerative Medicine 2013, Vol. 3, No. 3

11

Fig. (2). Histological staining and immunolabelings of fresh hAM (A, B, C, D, E, F) and cryopreserved hAM (G, H, I, J, K, L). (A, G) HES staining. (B, H) anti-human CD44 (Spring Biosciences, E17360). (C, I) anti-human CD73 (Epitomics, 5362-1). (D, J) antihuman type I collagen (Novotec, 20111). (E, K) anti-human type IV collagen (Novotec, 20411). (F, L) anti-human elastin (Novotec, 25011).

In 2004, Kim et al. [80] patented a cryopreservation medium composition for hAM containing Portulaca oleracea extract. It supplies effective nutrition to the hAM, reduces the possibility of a rejection reaction and causes fast epidermal regeneration and wound healing after operation. The

cryopreservation medium composition for treatment of ocular surface disease contains 49.5 to 85 % by weight of a medium, 14.5 to 50 % by weight of a cryopreservative and 0.0005 to 0.0015 % by weight of epigallocatechin gallate, 0.05 to 0.5 % by weight of Portulaca oleracea L. or 0.0005

12 Recent Patents on Regenerative Medicine 2013, Vol. 3, No. 3

to 0.5 % by weight of a mixture thereof as an antioxidant. The medium is selected among Eagle's Minimal Essential Medium, Roswell Park Memorial Institute medium (RPMI) 1640 and DMEM, and the cryopreservative is selected among glycerol and DMSO. Freeze Drying (Lyophilization), G-Irradiation, HyperDrying Lyophilization or freeze-drying is a preservation method that removes water from a tissue by sublimation. This results in the inhibition of destructive chemical reactions that can lead to tissue alteration. Lyophilized hAM can be stored at room temperature for a long period of time without deterioration and transportation is easy, thus resolving the main disadvantages of cryopreservation. Gajiwala et al. have described this technique in detail [48]. hAM was pasteurised at 60 °C after being cleansed, treated with 70 % ethanol and freeze-dried to remove 95 % of the moisture. G-irradiation has been widely used in the sterilization of numerous biomaterials or allografts. Appropriate doses of irradiation are effective against several bacteria, viruses and fungi. However, it has been found to affect the biological properties and tissue integrity (see [44] for references). A few studies have shown the efficacy of combining lyophilization and g-irradiation as a technique for sterilization of hAM. The packed and sealed hAM was sterilized by exposure to 25 kGy g-radiation in a cobalt 60 g-chamber unit or at an ISO-certified radiation plant. The freeze-dried and gsterilized tissue was stored at room temperature and protected from light for up to 6 months [81].

Gindraux et al.

Preservation Using Trehalose Even though lyophilized hAM retains most of the biological characteristics of non-preserved hAM, the freezedrying process affects some of its physical and biological characteristics; therefore it is not completely identical to native hAM. Nakamura et al. introduced the use of trehalose to stabilize and preserve the cellular membrane and proteins of lyophilized hAM [86]. Trehalose is a non-reducing disaccharide found in high concentration in many organisms that are capable of surviving almost complete dehydration. Its presence confers desiccation resistance on human cells. It has the ability to replace some of the water in the cell, thereby providing stabilization and protection of the amniotic structure and proteins during the lyophilization process. Although this method has shown preclinical effectiveness [86] and has been used to preserve other tissues/organs in various clinical applications, it has not yet been clinically applied to hAM for use in ocular surface reconstruction. 2. Effects and Limitations of the Preservation and Sterilization Techniques Preservation techniques, with or without an associated sterilization process, have been shown to influence the biological, histological and biophysical properties of hAM, particularly tissue thickness, tensile strength, elasticity and composition [69, 85, 87], biocompatibility [86], transparency [88], adhesion [41] and re-epithelialization properties [89]; growth factor release [90-92] and cell viability, especially of epithelial cells [43, 87, 90].

Clinical results showed excellent biocompatibility on the human ocular surface [82] or in other clinical indications Table 1. Gomes et al. modified this conservation method by sterilization of the hAM with ethylene oxide instead of gradiation [83]. A novel technique was recently elaborated involving initial hyper-drying instead of freeze drying. After being washed, the hAM was dried under consecutive farinfrared rays and microwaves at temperatures lower than 60 °C using a hyper-drying device. Final sterilization was performed as usual with 25 kGy g-radiation [25].

Briefly, it has been reported in the literature that there are no significant alterations observed in the tissue structure and extracellular components, such as collagen types I, III, IV, V and VII, fibronectin and laminin-5 between lyophilized hAM sterilized with 25 kGy of g-irradiation and cryopreserved hAM [81]. Niknejad et al. compared in in vitro studies, the adhesion properties of glycerol-cryopreserved and lyophilized hAM. They reported that the lyophilized hAM is a suitable matrix for cultivation of endothelial cells because lyophilization led to exposure of the basement membrane of the hAM [93].

PAA Sterilization

Growth Factor Release

PAA, a chemical in the peroxide organic family, is a standard sterilizing agent that is highly effective against bacteria, viruses, and spores due to its high oxidizing potential. It has been used in sterilization of an increasing number of biomaterials. It is ideal due to its segmentation to non-toxic residuals (acetic acid and oxygen peroxide) (see [44] for references). The clinical application and success of PAAtreated hAM in ocular surface reconstruction still remains to be proven.

One of the proposed mechanisms of action of hAM transplantation is the release of growth factors that facilitate corneal re-epithelialization and reduction of corneal scarring and inflammation. Studies on hAM have revealed the presence of various growth factors, including cytokines and signalling molecules, such as epidermal growth factor (EGF), transforming growth factor (TGF)- , - 1, - 2 and - 3, keratinocyte growth factor (KGF), KGF receptor (KGFR), hepatocyte growth factor (HGF), HGF receptor (HGFR), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), tumour necrosis factor (TNF), interferon (IFN), interleukin (IL)-4, IL-6, IL-8, natural inhibitors of metalloproteases, -defensins, and prostaglandins [1, 2, 93-96]. A recent publication reviewed the effects of the amnion and its cellular components within the inflammatory-fibrotic scenario and the factors described to date that could be involved in these immunomodulatory actions [97].

An alternative method adapted from bone tissue sterilisation consists in diluting 2 % PAA in 96 % ethanol under a negative pressure of 200 mbar and permanent agitation of the jar for 4 h to remove the air bubbles formed by the peracetic reaction [84]. First clinical experiences with PAA/ethanol sterilization and air-drying hAM allografts as a biological dressing for the treatment of corneal and conjunctival defects showed promising results (see [85] for references).

Clinical Uses of Human Amniotic Membrane

Recent Patents on Regenerative Medicine 2013, Vol. 3, No. 3

13

Higher levels of various growth factors (and potent mitogens) EGF, HGF, KGF, bFGF and of EGF receptor were found in hAM with epithelium versus without epithelium, indicating an epithelial origin for these growth factors [44, 94]. EGF is a potent mitogen for epithelial cell growth and its high level of expression could be an explanation for the promotion of ocular surface wound healing following transplantation. Thus, it can be suggested that after hAM transplantation, corneal re-epithelialization might be accelerated by KGF and HGF produced by the amniotic epithelium [94]. Fetterolf et al. showed, by immunohistochemical staining, the presence of some of these molecules in various amounts at different levels in the hAM [15]; but the presence of these growth factors is of the order of ng/mg in fresh tissue. There is a considerable paucity of data in this regard [98, 99]. Moreover, there are important variations in the rate of growth factors and proteins concentrations in hAM from different donors [98].

A few years ago, Hennerbichler et al. sought to understand how different storage conditions (with regard to media and temperature) may affect the viability of amnion [90]. They concluded that, independently of medium composition or temperature (liquid nitrogen or -80 °C), all the cryopreservation methods evaluated caused the same intense decline in hAM cell viability after thawing. More recently, the same group reported in vivo adhesion-reducing [105] and antifibrotic [106] properties of cryopreserved hAM in RPMI, DMSO and foetal calf serum / L-Glutamine / antibiotic– antimycotic solution [105] or human albumin [106] and stored at -80 °C. Moreover, they also described the use of an EZ4U cell proliferation and cytotoxicity assay (Biomedica, Austria), based on the principle of MTT to test the cell viability of hAM before xenotransplantation. We argue that these tests could easily become the standard for testing product safety by verifying cell viability before clinical implantation.

Several factors contribute to the biologic actions of hAM, which are regulated through IL-1, -4 and -6, EGF, bFGF, TGF- , - , KGF, NGF, endostatins, angiogenic factors and collagen I, II, III and IV, all of which were observed in cryopreserved hAM (see [37] for references). Hennerbichler et al. noted that several angiogenic growth factors and cytokines are removed during cryopreservation of hAM in DMSO at -80 °C [90]. Other authors have reported the presence of various growth factors in hAM preserved by lyophilization [81, 100] but this process causes greater reduction in total protein and growth factor content than cryopreservation [91], perhaps even to a significant extent [99]. Sterilization with grays seems to only marginally affect growth factor content in hAM [92]. Lopez-Valladares et al. reported that in nonpreserved, lyophilized and cryopreserved (at -80 °C or in liquid nitrogen) hAM, total protein amount, bFGF, HGF, KGF and TGF- 1 showed lower levels in samples from donors with higher gestational ages and donor ages [98]. Thus, if donor placenta has initially low levels of growth factors, treatment by lyophilization may be deleterious since it decreases the level of proteins.

As regards the preservation of cell viability, two patents have been reported:

Cell Viability

Immunological Properties

Literature regarding cell viability is sparse and findings sometimes contradictory, depending on the techniques used. This is a very important parameter because it is directly linked to the use of hAM in regenerative medicine and its possible immune response. Some conditions (e.g. tissue culturing, refrigerating or cryopreserving with DMSO) allow cell survival in hAM of about 40-90 % [43, 78, 101-103]. For example, at least 50 % of epithelial cells cryopreserved for 2 months in DMSO at -80 °C were still alive and had growth capacity in culture [78]. Regarding preservation in glycerol, the storage of hAM at 4 °C resulted in immediate cell death [90]; cryopreservation at -80 °C induced the loss of epithelial cell viability [43], and a highly concentrated solution abolishes hAM cell viability (see [44] for references). It seems that the viability of amniotic epithelial cells has little effect on the biologic properties of the hAM for ocular disorders; these properties are correlated with the integrity of its matrix. However, damaged epithelial cells may serve as a nutrient layer for migration and growth of conjunctival and corneal epithelial cells [104].

The immunological response to hAM transplantation is negligible, as demonstrated in several studies that have found no clinical signs of acute rejection in human volunteers [109, 110]. hAM seems to be immune-privileged tissue and several studies suggest that in vitro cultured epithelial and mesenchymal amniotic cells express small quantities of all HLA class Ia molecules (HLA-A, -B, -C, -DR) and express HLA class Ib (HLA-G, -E), which inhibit the cytotoxic potential of natural killer lymphocytes and synthesise anti-inflammatory and immunomodulatory molecules such as IL-10, TGF- and prostaglandin-E2 [78, 111, 112]. However, amnion epithelial cells do not express HLA class II antigens that may facilitate allogeneic transplantation [113]. The expression of Fasligand by epithelial and mesenchymal amniotic cells might exert an immunosuppressive effect by apoptosis. The literature concerning the immunogenicity of differentiated cells with respect to the features of the undifferentiated parental cells is very poor [114]. Recently, Tee et al. reported that immunogenicity and immunomodulatory properties were shown for hepatocyte-like cells derived from amniotic

-

In 2010, Zhou patented a hAM long-term preserving fluid (1000 mL of preserving fluid contains 10g-200g of thickening agent, 10g-50g of blood volume expansion agent, 5 mL-20 mL of amino acid, 5 mL-20 mL of antibiotics, 10 mL-50 mL of acid-base adjusting agent, 5 mL-30 mL of cell stabilizing agent, 5 mL-20 mL of cell nutrition constituents, and 860 mL-970 mL of culture solution) and the related preparation method [107].

-

In 2012, Tong patented a composite anti-freezing liquid (composed of DMEM, glycerol and DMSO at volume ratio of (4.5-5.5):(3.5-4.5):(0.5-1.5); and trehalose having a concentration of 0.05-0.15 mol/L) [108]. After pretreatment by the composite anti-freezing liquid, fresh hAM can be directly preserved in liquid nitrogen without refrigerating with a programmed cooling instrument, so as to simplify the procedure, save costs, shorten pretreatment time, reduce pollution, and maintain hAM activity in the long term.

14 Recent Patents on Regenerative Medicine 2013, Vol. 3, No. 3

epithelial cells [115]. They stated that in vivo studies on animal models of acute and chronic liver diseases will be needed to assess the functionality, immunomodulatory effects and immune responses. In a special issue, La Rocca and Anzalome suggested concerning these results that it would be interesting to follow the maintenance of the immunomodulatory features of undifferentiated stem cells also in the differentiated progeny of other stem cell types [114]. Clinical applications of hAM depend on the risk of immunogenicity, which is a function of the locality of transplantation and rejection dominant factors [116]. Moreover, several studies have indicated that the immunogenicity of cryopreserved hAM is lower than that of fresh hAM, because cryopreserved cells are expected to be nonviable [43, 78]. Since Hennerbichler et al. demonstrated that the viability of cells in hAM can be retained under certain conditions [90], it has to be taken into consideration that the cells on and in the tissue are still viable and possess certain properties, i.e. stem cell characteristics [117], expression of growth factors [118] and increased immunogenicity [43]. Regarding this fundamental parameter of immunity, two patents have been reported. Ramos et al. patented a method in 2004 for the preparation of immunologically inert hAM [119]. Briefly, after separation from the chorion and washing, the amnion is stretched in a perforated metallic sheet, dried with gauze and covered with gauze and a second perforated metallic sheet; the two edges of the sheet are folded in order to constitute the closed package. The closed package is sealed in a sterilizing sleeve and maintained for 96 hours at a temperature of 37 °C in an atmosphere containing 5 % CO2 . The hAM is then re-hydrated in physiological solution (0.9 % NaCl) and cryopreserved in culture medium containing 10 % DMSO. This immunologically inert hAM may be applied for the preparation of a product to be used as a skin substitute in 2nd or 3rd degree burns, as a nerve sleeve to guide regeneration of the peripheral nerves, as a cornea graft, in the reconstruction of the bladder and urethra, in the correction of cardiac malformations such as inter-auricular and interventricular communication and in the reconstruction of valvular leaflets. In 2012, Samson et al. [120] patented a cryopreservation procedure for a placental product that selectively depletes immunogenic cells (of at least one of immune cell type (e.g. CD14+ macrophages, trophoblasts, and/or vascular-tissue derived cells) and/or immunogenic factor that are otherwise present in a native placenta or hAM) and retains live therapeutic cells (including at least one or 2 or all of MSC, epithelial cells and fibroblasts) and/or retains therapeutic efficacy for the treatment of tissue injury. Such placental products contain viable therapeutic cells after thawing and are useful in treating patients with tissue injury, i.e. wound or burn, ligament and tendon repair or for engraftment procedures such as bone engraftment. III. USE OF HUMAN AMNIOTIC MEMBRANE FOR TISSUE ENGINEERING hAM is an attractive option for tissue engineering because it is easy to obtain, in unlimited quantities, with relatively inexpensive sampling and preparation processes. Furthermore contrary to available/commercialized scaffolds of

Gindraux et al.

animal origin or inert synthetic, hAM has the advantage to be from human origin with a small risk of infection or immune reaction. Thus, hAM can be used as a support matrix for regeneration, and it can promote re-epithelialization, reduce inflammation and suppress fibrosis. Because of the various structural components of hAM, such as collagen, laminin, fibronectin and vitronectin, adhesion and cell migration can be expected. These adhesion ligands activate signal pathways after interactions at cell surface receptors [121]. Regarding the use of hAM as a scaffold, Peyman [122] has patented compositions and uses of hAM with enhanced rigidity for biocompatible devices. To this end, hAM may be treated with a polymer and/or a crosslinking agent. The consistency-modifying component may be in an amount ranging from about 0.01 % w/w to about 99.99 % w/w. In one embodiment, the hAM is treated with radiation as the consistency-modifying component, resulting in a cross-linked hAM having enhanced rigidity in the absence of a chemical compound. 1. Decellularization Process Various techniques and reagents have been described to remove the epithelial cells from hAM in an attempt to produce a biological substrate on which various cell types can be seeded. There are currently many questions as to whether the intact or the denuded hAM is the better substrate for ex vivo expansion of epithelial cells for ocular surface construction (see [44] for references): Some authors have reported potential benefits of intact hAM; other groups have reported that denuded hAM can promote better cell proliferation and differentiation, better cell adhesion, as well as more uniform cell outgrowth compared to intact hAM. Hence, denuded hAM has been the preferred choice for ocular surface reconstruction up to now. Currently, there are several methods that have been described for denuding hAM: dispase ethylenediaminetetraacetic acid (EDTA), trypsin–EDTA, urea, ethanol, thermolysin, hypotonic buffer, sodium dodecyl sulfate (SDS) and nuclease methods. Each method has been shown to have different effects on the membrane structure and its biological properties. Ideally, the de-epithelialization method should be efficient and should not alter the structural integrity (in particular, of the basal membrane) or biological function (see [44] for references). The removal of epithelium eliminates nearly all important growth factors [92]. Therefore, determining which hAM preparation, intact or denude, is appropriate for tissue engineering depends on other conditions, including the type of cell or tissue being used. For example, Wilshaw et al. showed in in vitro studies that denuded hAM was capable of supporting the attachment and proliferation of primary human fibroblasts and keratinocytes; the viability of the cells was maintained for up to 4 weeks [123]. Other authors reported, in a rat model, that the extracellular matrix of denuded hAM is an effective conduit for peripheral nerve regeneration and that the hAM is a biodegradable scaffold with unique biochemical and mechanical characteristics for nerve regeneration [124, 125]. Recently, Guo et al. described a simple method to produce denuded hAM with preserved biomechanical properties and a favourable adhesion potential [126].

Clinical Uses of Human Amniotic Membrane

Recent Patents on Regenerative Medicine 2013, Vol. 3, No. 3

15

Some decellularization treatments have resulted in patents, see Table 3, or have been used to produce hAM for commercialization, see Table 4.

failure. The transplanted cells are thought to regenerate the ocular surface by restoring an intact transparent corneal epithelium.

In 2002, Xue [127] patented an engineered scaffold of tissue prepared from hAM whose epithelial cells have been removed. The process for removing these cells includes immersing in 0.2-5 % trypsin solution at 37 °C for 20-25 min and stirring while flushing. Its advantages are the absence of immunoreaction and the simplicity of the process.

This ex vivo culture has also been performed in other settings, namely:

In 2009, Kim et al. [128] patented a novel preparation method for decellularized hAM that comprised a first step of freezing hAM; a step of thawing; and a step of centrifugally separating the thawed hAM. The hAM is selected from among human, pig, sheep, goat, horse, alpaca, mouse, rat, or cow and improves the healing effect on chemical burn to the cornea. Also in 2009, Guhathakurta et al. [129] patented a method for treating hAM that consists in decellularization (in deoxycholic acid solution), reorganisation of the collagen matrix with heparin in glucose solution and cross linking (physico-chemical photooxidation with methylene blue and UV radiation). This makes the hAM adaptable for adhering to the wound area and enhancing growth of neighbouring tissue, allowing the foreign membrane placed on the wound area to merge with the neighbouring skin. The treated hAM that actively controls the whole process of skin regeneration can be used as dressing, as a graft and for transplantation. In 2010, Cong et al. [130] patented a method that comprises the following steps: soaking and disinfection of the fresh hAM by tobramycin sulfate injection; digestion using trypsin-EDTA and light scraping of the epithelial surface. Denuded hAM is then tiled in culture plate holes for fixing and dry-posting, coating by special coating liquid for the denuded hAM; sucking and then drying the coating liquid to obtain the carrier bracket of the tissue engineering artificial corneal endothelium. In 2012, Han et al. [131] patented an acellular-nucleic acid degradation method that comprises soaking of hAM in, first, a hypotonic tris(hydroxymethyl)aminomethane buffer solution at 4 °C for 16 h; second, a tris-buffered saline-SDS solution with rotation at 25 °C for 24 h; and third, a nucleic acid degradation solution at 37 °C for 3 h. After being freeze-dried, packaged, irradiated and sterilized, the acellular hAM can be preserved at normal temperature for a long time. 2. Human Amniotic Membrane as a Scaffold for Tissue Engineering Cultivation and seeding of epithelial cells on an amnion scaffold is a frequently used method for ocular surface and skin reconstruction. Ocular Surface Reconstruction HAM has been used as a culture substrate for ex vivo cultured limbal epithelial stem cell transplantation (see [96] for references). The use of hAM for this purpose in humans was first described by Schwab in 1999. The process involves the transfer of autologous or allogeneic human limbal epithelial cells cultured on hAM onto the eyes of patients with severe damage and scarring from limbal epithelial stem cell

-

in clinical studies with human conjuctival epithelial progenitor cells [132] or with oral mucosal epithelial cells [133];

-

in animal studies with human keratocytes isolated from central corneal buttons [134] or human retinal pigment epithelial (RPE) cells [135].

There are marked variations in the methods used to prepare hAM prior to culture of limbal or conjunctival epithelial stem cells (see [96] for references). For example, cryopreserved hAM is used as a substrate for the ex vivo expansion of limbal and conjunctival stem cells [136]. G-irradiated lyophilized hAM has been used successfully as a scaffold for conjunctival epithelial cell growth following bare sclera excision of pterygium [82]. In 2001, Tsai [137] patented a method and graft for treating epithelial stem cell and limbal stem cell deficiency in, the reconstruction of cornea, for example. Transplantation of epithelial stem cells, cultured ex vivo on specifically treated hAM, yields a surgical graft having expanded epithelial stem cells. The source of the epithelial stem cells can be a very small explant from healthy autologous and allogeneic tissue biopsy; the hAM is treated such that its extracellular matrix is maintained, but its cells are killed. In 2006, Binder et al. [138] patented a composition, i.e. cryopreserved hAM and a plurality of RPE cells or RPE equivalent cells present at the hAM for implantation in the subretinal space of an eye. hAM may be intact, denuded (without epithelium), or otherwise treated. The composition does not elicit immunological reactions to allo-antigens or to RPE specific auto-antigens. Skin Reconstruction With regard to use of hAM for skin reconstruction, Yang et al. reported the use of denuded hAM seeded with keratinocytes and grafted in an animal model of a full-thickness wound [139]. Redondo et al. reported the use of hAM seeded with melanocytes and transplanted onto distinct achromic lesions in patients with stable vitiligo [140, 141]. In 2012, Xia et al. [142] patented a hAM microcarrier capable of simulating a niche microenvironment for the growth of epidermal stem cells in vitro and used as a dermal scaffold for the construction of a skin substitute. hAM is subjected to a repeated freeze-thawing and DNA enzyme digestion method to remove amniotic cells, and is further subjected to freeze-cracking to be prepared into the hAM microcarrier able to support the in vitro culture and amplification of epidermal stem cells. Vascular, Orthopaedic, Oral and Maxillofacial, and Urothelial Tissue Engineering As a potential approach for vascular tissue engineering, cultivation of human corneal endothelial cells on a denuded hAM scaffold has also been reported in a rabbit model [143];

16 Recent Patents on Regenerative Medicine 2013, Vol. 3, No. 3

and cultivation of porcine vascular endothelial cells on a cryopreserved hAM in in vitro studies [144]. Denuded hAM has been investigated in the rabbit as a carrier of rabbit chondrocytes, and it has been suggested that the hAM can serve as a carrier matrix for cartilage regeneration [145]. These data were confirmed by in vitro studies performed: (i) with human chondrocytes seeded on denuded hAM [146], (ii) with a new 3D scaffold synthesized from hAM and fibrin and seeded with bovine chondrocytes [147], and (iii) with rabbit MSC seeded on a 4 °C saline stored overnight, hAM was shown to be able to differentiate into chondrogenic cells [148]. Similarly, denuded hAM as a scaffold demonstrates promising potential in facilitating in vitro osteogenic differentiation by modulating an appropriate environment for human apical papilla cells (APCs). Authors demonstrated that denuded hAM could potentiate the induction effect of osteogenic supplements and promote osteogenic differentiation of human APCs [149]. In original works, Lindenmair et al. reported in a proof of concept study, that viable hAM in toto – with its sessile stem cell components – possesses potential as grafting material for bone regeneration [150]. Under in vitro osteogenic conditions, the intact viable hAM became mineralized and expressed markers of early and late osteoblastic differentiation, including alkaline phosphatase, RUNX2, Bone Morphogenetic Protein (BMP)-2, BMP-4 and osteocalcin. Osteocalcin expression was demonstrated also in non-mineralized areas also containing proliferating cells, which may indicate that some cell regions in the hAM lag behind in the differentiation process, while others already show signs of mineralization together with extracellular matrix deposition. The same group [151] patented this proof of concept. Expanding on this, Laurent et al. are investigating whether osteodifferentiation of intact hAM induces immunity acquisition by amniotic cells. This work consists in observing immune reaction after 1, 2, 4 and 8 weeks of implantation of osteodifferentiated hAM in subcutaneous murine model. First results show no significant inflammatory tissue (Laurent et al. submitted). The options for hAM in oral and maxillofacial tissue engineering are manifold. hAM scaffold has served as a cultured stratum for many cell types, such as: -

in ocular surface reconstruction in the rabbit, with rabbit oral epithelial cells seeded on denuded hAM [152];

-

on the latissimus dorsi muscle in the rabbit with rabbit oral keratinocytes seeded on denuded lyophilized and sterilized hAM [153];

-

in oral mucosal defects in the rabbit with oral mucosal epithelial cells seeded on hAM [154];

-

in in vitro studies with murine foetal submandibular gland explants cultured on denuded or cryopreserved hAM scaffolds [155].

Finally, hAM has been proven to be suitable for urothelial tissue-engineering applications. First, Koziak et al. published clinical data on reconstructive surgery of a male urethra using hAM [156]. Subsequently, Shakeri et al. reported uroepithelium repair in rabbits [157]. Sartoneva et al. tested in in vitro studies the culture of human urothelial cells on denuded hAM, but with questionable results [158]. A process for establishing a highly differentiated urothelium on

Gindraux et al.

connective tissue of intact hAM has been patented by Kreft et al. [159]. 3. Human Amniotic Membrane as a Carrier of Bioactive Molecules In 2004, Zhang [160] patented reconstituted and recombinant tissue membranes and methods for pharmaceutical delivery of bioactive molecules. This recombinant tissue contains one or more recombinant expression vectors that are exogenous to the membrane and capable of expressing bioactive molecules, such as growth factors, anti-microbial proteins, anti-inflammatory protein, protease inhibitors or hair growth promoting factors, and can be used for in situ delivery of therapeutic proteins to a host in the treatment of disorders such as chronic wounds and dermatologic or ocular surface diseases. In 2006, Fang [161] patented a drug hAM containing an exogenous cell growth factor. The preparation comprises immersion of the hAM into a storage liquid with exogenous cell growth factor and storage at ultra-low temperature or drying. In 2008 Dua et al. [162] patented methods of processing hAM to generate a substantially “growth factor-free” membrane and to enrich it with specific and quantified levels of growth factors or other desirable membrane enriching molecules or compounds. Recently, Tseng et al. [163-166] filed four patents on specific biological components that have been found to exert a number of useful effects in mammalian cells, including modulating TGF- signalling, apoptosis, and proliferation of mammalian cells, as well as decreasing inflammation in mice. These components can be obtained commercially, or can be prepared from biological tissues such as placental tissues (amniotic membrane pieces, extracts, jelly, stroma, ...). The compositions can be used to treat various diseases, such as wound healing, inflammation and angiogenesisrelated diseases. Apart from these patents, Mayer et al. reported in an in vitro setting, that cryopreserved hAM can successfully be incubated with bevacizumab and serve as a carrier for antiVEGF drugs to provide constant VEGF blockade and to avoid neovascularization, a common complication of corneal ulceration [167]. IV. HUMAN AMNIOTIC MEMBRANE MANUFACTURED PRODUCTS AND ECONOMIC ASPECTS 1. Products and Future Markets Dua et al. describe how J. F. Batlle and F. J. Perdomo presented a paper in 1992 entitled “Placental membranes as a conjunctival substitute” to the annual congress of the Dominican Society of Ophthalmology in the Dominican Republic. In this presentation, Batlle traced the use of “Soviet tissues” as allotransplants (dispensed as a sheet spread on a carrier or in a vial) in conjunctival, tarsal, orbital, and tendon surgery. This first commercialized hAM dubbed “The Russian allotransplant” was later proven to contain human foetal membranes [42].

Clinical Uses of Human Amniotic Membrane

Since then, hAM transplantation has clearly gained a pivotal position in the clinical management of ocular surface disorders. Advanced tissue stabilization and preservation techniques have caused a resurgence in the use of this material in the treatment of chronic or non-healing dermal wounds. The Purion® process (MiMedx Group, Inc, Kennesaw, GA, USA) has created a stable, easily stored, and easily transported material that is becoming an increasingly common application in the treatment of various types of wounds. This tissue processing technology uses a combination of procedures designed to select low-risk patients, cleanse the membrane of bioburden, gently dehydrate the material, and sterilize the prepared product. Initially marketed to the ophthalmology community, hAM allografts made using this process have been successfully employed more than 45,000 times in the treatment of eye injuries, burns, and for reparative eye work such as correction of pterygium. More recently, the success of this material as an allograft has been extended to the general treatment of cutaneous wounds [15]. The future of hAM transplantation is promising, with continued technological advancements in tissue engineering. Innovations such as AMX, Prokera®, and Acelagraft™ have benefitted from the previously described hAM preparation techniques and have made access to hAM easier than ever before, see Table 4. For example, AMX is a topical application of hAM extracts, currently only available in Europe. Prokera®, devised by Tseng, comprises hAM attached to a soft contact lens-sized conformer for easy insertion [168]. Acelagraft™ is a denuded hAM that has been lyophilized and sterilized with g-irradiation and that can be shipped and stored at room temperature [169]. However, our understanding of the best methods of hAM preparation and its mechanisms of action is still far from complete, and may remain so for the foreseeable future. In a comparison of efficacy study [169], Bhatia et al. compared Amniograft® and Prokera® (hAMs that are frozen, with intact cellular structure, associated growth factors, and cytokines) to Acelagraft(TM)™. Results showed that Acelagraft™ has demonstrated potential as a wound healing product in ongoing studies for the treatment of acute and chronic ulcers, even with all cells and related factors removed. Lim et al. compared the biological and ultrastructural properties of Acelagraft™ with cryopreserved hAM and demonstrated the feasibility of Acelagraft™ transplant in a case of chronic ocular surface disease, although its biological properties have not been well characterized [100]. The future of hAM transplantation also will undoubtedly involve its application in new fields, for example in orthopaedic, spinal, urological, gynaecological, and oral and maxillofacial surgeries, summarized in Tables 3 and 4. Some of these manufactured hAM have been tested in clinical trials elsewhere for new indications. For example, Amniograft® has been tested for periodontal soft tissue healing [37]; BioCover™ for gingival recession [27] and Amnioclear® for the repair of posterior tibial and Achilles tendons [28]. Finally, a new horizon for hAM could be research into sutureless transplantation in the ocular field, as previously reported [170-173]. For example, Prokera® has also been

Recent Patents on Regenerative Medicine 2013, Vol. 3, No. 3

17

used to cover the corneal surface via a US FDA-approved medical device, termed without sutures [40]. 2. Economical Aspects Few publications have reported economical aspects of hAM uses: -

In 2007, Pesteil et al. stated that the additional cost associated with the use of hAM to treat arterial or mixed origin ulcers corresponded to 2090 Euro for 27 weeks of treatment [61]. One membrane was used for each patient per week; the price of a cryopreserved hAM was 78 Euro.

-

In 2010, Velez et al. reported in a clinical study that with regard to dental implants, the placement of cryopreserved hAM was not cost effective [37].

-

In 2011, Gutierrez-Moreno et al. analysed the literature on the safety and efficacy of hAM grafting and compared the cost of currently available autografts, hAM, and biocompatible skin substitutes to promote tissue repair in venous ulcers [57]. They reported that only one study addressing safety and efficacy was identified. Cost-minimization analysis showed that autografts are always the least expensive option (1053 Euro compared with 1825 Euro for hAM and 5767 Euro for biocompatible skin grafts). However, at 6 months, hAM would have cost 6765 Euro less than the use of biocompatible skin substitutes. The cost of a graft in these authors’ institution (“servicio de Dermatología, Hospital Clínic, TSF, Universitat de Barcelona, Barcelona, Spain”) was 425 euros.

CURRENT & FUTURE DEVELOPMENTS The reconstruction of ocular surfaces with hAM has become a well-established, routine procedure since the 1990s. Results of past and current clinical trials performed in wound healing will enable this technique to become routinely applied in this field. Clinical applications of hAM have been less frequently reported in oral and maxillofacial, ear-nosethroat, gynaecological and orthopaedic surgeries to date, but results in this area are encouraging and clinical trials are ongoing. These new clinical indications are based on the regenerative potential of amniotic (stem) cells, and their ability to differentiate, and address the pathology. This will imply: (i) improvements in preservation/sterilization techniques to maintain cell viability, especially after long term storage, (ii) precise studies of the immunological properties possibly acquired by amniotic cells during their cell differentiation, and (iii) complete quantification of released growth factors. Another future avenue of hAM research will be its use as autograft in the case of prenatal pathological diagnosis. This will involve scheduled early delivery and a preservation process different from hAM allografts. This review shows that processed hAM can easily be used as a “simple scaffold” or as a carrier to deliver biological molecules, and many commercially available products are to be found in this area. Existing processed products will

18 Recent Patents on Regenerative Medicine 2013, Vol. 3, No. 3

certainly be improved in the future to render these inert scaffolds bioactive, thereby making these “simple scaffolds” functional. Finally, a very wide body of literature exists on MSC derived from hAM, and this tissue constitutes a very interesting source of allogenic MSC with all the attractive properties of foetal/perinatal tissue in comparison to adult allogenic MSC derived from bone marrow or adipose tissue. These new avenues of research, such as new indications for hAM allograft, functionalization of inert membranes, use of hAM for autografts, and studies on MSC derived from hAM, will most certainly lead to a plethora of new patents in this exciting and blossoming domain. CONFLICT OF INTEREST

Gindraux et al. [12] [13] [14] [15] [16]

[17] [18]

The authors confirm that this article content has no conflicts of interest. [19]

ACKNOWLEDGEMENTS Thanks to Martine Melin and Aurélie Nallet (Novotec, Lyon, France) for histological studies. The authors also thank Fiona Ecarnot for editorial assistance. REFERENCES [1] [2]

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