Regenerated Silk Fibroin Scaffold and Infrapatellar Adipose Stromal ...

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Jun 8, 2011 - Massimo Faustini, Ph.D.,5 Mario Marazzi, M.D.,3 and Maria Luisa Torre, Ph.D.1. Articular cartilage has limited repair and regeneration potential ...

TISSUE ENGINEERING: Part A Volume 17, Numbers 13 and 14, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tea.2010.0636

Regenerated Silk Fibroin Scaffold and Infrapatellar Adipose Stromal Vascular Fraction as Feeder-Layer: A New Product for Cartilage Advanced Therapy Theodora Chlapanidas, Ph.D.,1 Silvio Farago`, Ph.D.,2 Federica Mingotto, Biotech.D.,3 Francesca Crovato, Eng.D.,3 Marta Cecilia Tosca, Biotech.D.,3 Barbara Antonioli, Biol.D.,3 Massimo Bucco, Ph.D.,1,* Giulia Lucconi, Pharm.D.,1 Alessandro Scalise, M.D.,4 Daniele Vigo, Ph.D.,5 Massimo Faustini, Ph.D.,5 Mario Marazzi, M.D.,3 and Maria Luisa Torre, Ph.D.1

Articular cartilage has limited repair and regeneration potential, and the scarcity of treatment modalities has motivated attempts to engineer cartilage tissue constructs. The use of chondrocytes in cartilage tissue engineering has been restricted by the limited availability of these cells, their intrinsic tendency to lose their phenotype during the expansion, as well as the difficulties during the first cell adhesion to the scaffold. Aim of this work was to evaluate the intra-articular adipose stromal vascular fraction attachment on silk fibroin scaffold to promote chondrocytes adhesion and proliferation. Physicochemical characterization has demonstrated that three-dimensionally organized silk fibroin scaffold is an ideal biopolymer for cartilage tissue engineering; it allows cell attachment, scaffold colonization, and physically cell holding in the area that must be repaired; the use of adiposederived stem cells is a promising strategy to promote adhesion and proliferation of chondrocytes to the scaffold as an autologous human feeder layer. Introduction

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ilk is produced by a wide variety of insects and spiders: the most common one is produced by the silkworm of Bombyx mori; the B. mori cocoon is composed of two proteins: fibroin (75%) and sericin (25%). Fibroin contains principally residues of glycine (46%), alanine (29%), and serine (12%), whereas the most representative aminoacids in sericins are serine (37%), glycine (17%), and aspartic acid (16%).1 Silk fibroin shows different molecular conformations: Silk II selfassembles to form an anti-parallel b-sheet structure, dominant in fibers; such structure is stable and insoluble in water. Silk I is characterized by a mixture of random coil, a-helix, and b-turn structures, and is metastable, water soluble, and dominant in fibroin solutions. The cristallinity degree of fibroin depends on the b-sheet structure content. Fibroin is synthesized in the posterior region of B. mori silk gland as Silk I. When silk fibroin undergoes the spinning process in the anterior region of the gland, it changes its conformation in Silk II.2 Besides its widespread employ in textiles, silk is also known for its use in several biomedical fields as suture

material and as scaffold constituent in tissue engineering. This is due to unsurpassed mechanical properties, as traction resistance, its biocompatibility, and biodegradability. Over the past few years, numerous studies have explored the potential of fibroin-based biomaterials in various forms, including films, hydrogels, nonwoven mats, nets or membranes, and three-dimensional (3D) porous sponges.3 The in vitro engineered articular cartilage for transplantation has been in clinical use from many years: autologous chondrocytes gained from small cartilage biopsies and in vitro propagated were transplanted and good long-term results are achieved with this technique.4,5 During proliferation in monolayer conditions, however, chondrocytes dedifferentiate and cease producing the mechano-biologically indispensable matrix proteins collagen type II, aggrecan, and others. Therefore, a new operation procedure of matrix-associated autologous chondrocyte transplantation has been developed: cells are seeded on scaffold to improve extracellular matrix production, to prevent chondrocyte dedifferentiation or to promote chondrocyte re-dedifferentiation. Moreover, scaffolds are useful for the immobilization of the cells, for a

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Dipartimento di Scienze del Farmaco, Universita` di Pavia, Pavia, Italia. Stazione Sperimentale per la Seta, Milan, Italy. 3 Struttura Semplice Terapia Tissutale, Azienda Ospedale Niguarda Ca’ Granda, Milan, Italy. 4 Dipartimento di Scienze Mediche e Chirurgiche, Universita` Politecnica delle Marche, Ancona, Italy. 5 Dipartimento di Scienze e Tecnologie Veterinarie per la Sicurezza Alimentare, Universita` di Milano, Milan, Italy. *Current affiliation: Bioscience Institute, Falciano, Repubblica di San Marino. 2

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1726 broader distribution of the cells in the defect, and for facilitating the handling during surgery: the scaffold architecture, structure, and composition can dramatically influence cell adhesion, distribution, proliferation, and differentiation cell– scaffold graft.6 The use of mesenchymal stem cells has recently been proposed to improve this therapeutic approach since they represent a population of multipotential cells able to regenerate not only cartilage but also bone, and are easier to obtain compared to chondrocyte cells. Autologous bone marrowderived mesenchymal cells prove to be as effective as chondrocytes for articulage repair, offering undoubtedly clinical advantages as normal articular cartilage is not damaged to recover the bioptic tissue.7 A new prompt in this direction was given by the findings that adipose tissue had been identified as a source of mesenchymal stem cells. Adipose stromal vascular fraction (SVF) contains an etherogeneous pool of cells as endothelial, smooth muscle cells, pericytes, fibroblasts, macrophages, preadipocytes, and adiposederived stem cells (ADSCs).8 Independent research groups have studied yield and proliferation/differentiation potential of these cells with respect to adipose collection sites.9,10 Autologous mesenchymal stem cells are also an optimal support for nucleus pulposus cells, to enhance intervertebral disc regeneration, to reduce the risk of rejection, and to promote the scaffold colonization or extra-cellular matrix production.11,12 For cell therapy and regenerative medicine use, stem cells are separated from their natural niche, manipulated, and transplanted in a different site: this procedure assumes that cells are equivalent, and they can substituted each other without altering development.13 In our knowledge, this assumption is actually not yet demonstrated and we cannot know if all the mesenchymal stem cells can influence tissues positively during regeneration, but perhaps negatively toward the pathogenesis of cancer and metastasis, as recently reported by Becerra et al.13 Recently, Wickham and colleagues14 have demonstrated that infrapatellar fat pad (IFP) of the knee, also known as Hoffa’s body, could be a new source of multipotent stromal cells. The IFP is an intra-articular adipose tissue that occupies the area between the patellar ligament and the infrapatellar synovial fold of the knee joint. In this article the use of intraarticular mesenchymal stem cells was proposed for the cartilage regeneration: ex vivo reproduction of the physiological microenvironement could mimic the natural niche and promote the tissue regeneration. The SVF was isolated from IFP adipose tissues and employed to promote chondrocyte colonization of silk fibroin scaffold. SVF was also isolated from subcutaneous tissue for each subject as a control. Silk fibroin was characterized in terms of amino acid composition, mass weight distribution, molecular conformation, and differential scanning calorimetry (DSC) analysis. Cocultures of chondrocytes and adipose SVF were performed and a morphological investigation was carried out. A commercial hyaluronic acid scaffold was used as a control. Materials and Methods

CHLAPANIDAS ET AL. trolled microbial environment. The same institute provided also silk fibroin extracted from the anterior region of B. mori glands. All the cocoons were degummed in autoclave at 120C for 1 h (40 mL of water/g of cocoons), to eliminate sericine from silk fibers. Cocoons were rinsed three times with water at 60C, dried at room temperature for 36–48 h, cut in small pieces, and treated with a solution of Ca(NO3)2 (75% w/v) in methanol under stirring at room temperature, to obtain a raw fibroin solution, as reported by Mathur et al.15 After filtering using a vacuum filtration device equipped with a 0.1 mm pore size PTFE membrane, the solution was dialyzed against distilled water using a cellulose membranes (cut-off 12,000 Da) at temperature of 25C. The final concentration of silk fibroin aqueous solution was 2% w/v. Fibroin amino acid composition A sample of silk fibroin solution was liofilized, and the amino acid composition was determined by HPLC analysis (HPLC Waters–Separation Module: alliance 2695, Pump Mod. 510, Pump control module E-sat/in, heater 410, Photodiode array detector2996, injection 77251, differential refractometer, Picotag workstation). Samples were treated with HCl 6N at 105C for 24 h, added of a-aminobutyric acid (used as internal standard), and then filtered with 0.45 mm membrane. Ten microliters of this solution was dried (Picotag vacuum system) and then solubilized in 50 mL of HCl 20 mM. Samples were derivatized with Waters AccQ-Fluor Reagent kit at 55C for 30 min, and chromatographic separation was carried out using an AccQ-tag column (Waters). The mobile phase consisted of borate buffer, acetonitrile, and water (flow rate 1 mL/min) and the injection volume was 10 mL. Each determination was carried out in duplicate. Results are reported as w/w percentage of amino acid. The amino acid composition was evaluated also for fibroin collected from B. mori glands, after direct collection from B. mori glands, and treated at the same way. Silk fibroin mass weight distribution A sample of silk fibroin solution was treated ready-made, and the mass weight distribution was determined by HPLC (see above). Chromatographic separation was carried out using a Shodex column. The mobile phase consisted of phosphate buffer pH 7.2, 0.15M KCl, and 5 M urea, and the injection volume was 100 mL. Each run lasted 30 min with a column temperature of 32C. The mass weight was evaluated with external standard method: a calibration curve was obtained considering eight standard molecules as reported in Table 1. The mass weight distribution was evaluated also for the silk fibroin solution after direct collection from B. mori glands, and treated in the same way. Preparation of silk fibroin scaffold Silk fibroin solution was gently poured into molds, and the preparations were freeze-dried. Scaffolds were immersed in ethanol for 12 h to induce a complete conformational transition from silk I to silk II and then dried at room temperature.

Fibroin solubilization

Fourier Transform Infrared Spectroscopy

Cocoons of B. mori were supplied by the Zoological Institute of Padua (Italy); breeding was carried out under con-

Before and after ethanol treatment, scaffolds were analyzed by Fourier Transform Infrared Spectroscopy (FTIR) on

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Table 1. Standard Calibration Curve for the HPLC Analysis of Molecular Weight Distribution Standard Tyrosinase Alcohol dehydrogenase Bovine serum albumin Albumin Carbonic anhydrase Myoglobin Ribonuclease A Cytochrome C

Molecular weight (Da)

Log molecular weight

Retention time (min)

669,000 150,000 66,000 64,600 29,000 18,800 15,700 12,400

5.825 5.176 4.820 4.810 4.462 4.274 4.196 4.093

12.766 14.686 15.910 17.053 19.244 20.455 21.104 21.762

y = - 5.4662x + 43.602, R2 = 0.9493, calculated on the logarithm of molecular weight.

a Bruker Alpha-E spectrometer equipped with a MIRacle attenuated total reflection Diamond crystal cell in reflection mode. Background measurements were taken twice with an empty cell and subtracted from the sample readings. The FTIR spectra in the absorbance mode were obtained in the spectral regions of 500–3500 cm - 1. Each spectrum of the samples was acquired by accumulation of 32 scans with a resolution of 4 cm - 1. Native silk fibroin was used as a control. DSC Analysis Native silk fibroin and scaffolds before and after ethanol treatment were analyzed by DSC Mettler TA30. Thermal scanning was carried out on 3 mg of each sample in nitrogen (temperature range: 10C–500C, rate: 10C/min).

Cell culture Regenerated silk fibroin scaffolds were cut to obtain pieces of 2 · 1 · 0.5 cm. Intra-articular SVF was seeded on the surface of the regenerated silk fibroin scaffolds (0.5$106 cells/cm2), incubated in DMEM, 10% fetal bovine serum, 1% penicillin/ streptomycin, and 1% Amphotericin B for 7 days (37C, 5% CO2); after this time, chondrocytes were seeded on the same scaffold (cell density: 0.5$106 cells/cm2); co-culture lasted 21 days from the start of SVF culture. At the same time, chondrocytes were seeded on silk fibroin scaffolds for 21 days in the same conditions. A commercial esterified hyaluronic acid scaffold (HYAFF, Fidia Farmaceutici) was employed as a control and cells were seeded in the same conditions. At 7, 10, and 21 days, samples were fixed with 35% formaldehyde in PBS medium, dehydrated by alcohol scale, and embedded in paraffin. Sections were stained with hematoxylin/eosin.

SVF isolation For this study 24 informed subjects were enrolled (7 men and 17 women; mean – SD age, 63.2 – 16.3; age min–max, 15–87 years; mean – SD body mass index [BMI], 28.1 – 4.0; BMI min–max, 20.99–38.45). Adipose tissue samples were resected during surgery for knee articular diseases in both subcutaneous and intra-articular layers. In the operating room, after sampling, biopsies were suspended in phosphatebuffered saline (PBS) with penicillin/streptomycin 1%, put into a sterile box, and forwarded to the laboratory at a temperature of 4C. Samples were digested with 0.02% collagenase (Sigma) in PBS plus 1% penicillin/streptomycin. SVF was centrifuged and washed twice with PBS medium, as reported by Zuk et al.16 Harvested stromal cells were counted under the microscope, and their viability was assessed by the trypan blue exclusion technique. For each sample, recovered cell number, percentage viability, cell yield (total cell number/mL adipose tissue), and live cell yield (live cell number/mL adipose tissue) were screened. All samples were counted in triplicate by counting at least 200 cells per slide. Knee articular cartilage was harvested from cadaver donors, treated with 0.05% trypsin (Sigma-Aldrich) for 30 min, centrifuged, and washed. Tissue was digested with collagenase 200UI (Sigma-Aldrich) overnight. Cells were recovered and suspended in Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum, 2% penicillin/streptomycin, and 2% L-glutamine into T75 flasks (500,000 cells/flask) and incubated at 37C. Once the adherent cells reached sub-confluence, they were treated with 0.05% trypsin, resuspended in DMEM, and counted.

Immunofluorescence staining Immunofluorescence investigation was performed by laser confocal microscopy. Paraffin-embedded samples were cut into 5-mm-thick sections and mounted on immunofluorescence microscope slides. The sections were treated with a 1% Triton X-100 solution in PBS medium; to block aspecific binding sites, samples were treated with 2% bovine serum albumin in PBS medium and left to stand for 30 min at room temperature. After this period, samples were treated (37C for 60 min in light proof conditions) with primary antibody against CD90 (mouse-antihuman, 1:200 dilution, Chemicon),

FIG. 1. Aminoacid percentual composition (w/w) of gland fibroin and fibroin solution.

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CHLAPANIDAS ET AL.

FIG. 2. Chromatogram of silk fibroin recovered from gland (a line) and after extraction from cocoons (b line).

CD34 (class I MAb clone, 1:400 dilution; Chemicon), and Collagen Type II (MAb clone, 1:200 dilution; Chemicon). Primary antibodies were diluted in PBS medium with bovine serum albumin 1%. The samples were further treated with the secondary dye-coupled antibody, Goat anti Mouse IgGRhodamine (TRITC) conjugate (1:200 dilution; Chemicon). The slides were then treated with Vectashield liquid mounting media. Slides were observed under a Leica confocal fluorescence microscope with oil immersion 40 · objective, 1.32 NA aperture. For each field, three confocal planes were acquired, and the elaboration of planes was performed by FluoView (Leica) software. Images were stored as single TIFF 24-bit with three distinct channels for red, green, and blue. Scanning electron microscopy Before cell culture, regenerated silk fibroin scaffolds were analyzed using a scanning electron microscope ( JEOL JSM6380LV) operating at low vacuum degree, 20 kV, retrodiffused electron signal. Silk fibroin scaffold and hyaluronic acid scaffold surfaces were observed also after 7 days of stromal cell culture. Samples were treated with 2% glutaraldehyde for 30 min at room temperature, then 2% glutar-

FIG. 3. Fourier Transform Infrared Spectroscopy spectra of native silk fibroin, and scaffolds before and after treatment with ethanol.

aldehyde and cacodylate buffer 0.1 M for 30 min at 4C, and finally washed with cacodylate buffer 0.1 M. Samples were dehydrated using a graded ethanol series (from 30% to 100%), freeze-dried, critical point dried, and sputter coated with gold. The effect of ethanol treatment on silk fibroin scaffold was analyzed in the same operative conditions. Statistical analysis To test the influence of gender, collection site, age, and BMI on SVF cell yield and live cell viability, the analysis of covariance (ANCOVA) was applied, considering gender and collection site as fixed factors, and age and BMI as covariates. Data were previously analyzed using Levene’s test to assess the equality of variance in different samples (omoscedasticity). Between covariates, a multiple linear regression model was used to value the correlations between independent variables. Results were also evaluated with Wilcoxon test, regardless of subject variables (age, gender, and BMI). Results Fibroin amino acid composition is reported in terms of w/w percentage for fibroin solution and gland fibroin (Fig. 1). Results indicate that the main aminoacids are glycine,

SILK FIBROIN SCAFFOLD FOR TISSUE ENGINEERING alanine, and serine; the percentage of Gly is 47.70% in silk fibroin from glands and 45.07% after solubilization; the values for Ala are 25.81% and 28.11% (glands and solubilized, respectively) and for Ser are 11.14% (glands) and 11.38% (solubilized). The mass weight distribution was evaluated for silk fibroin recovered from glands and after extraction: silk fibroin from cocoons yielded a peak at the same retention time with respect to silk fibroin from gland (Fig. 2), and from the calibration curve, a molecular weight of about 230 kDa was calculated for the maxima of both elution peaks. Regenerated aqueous silk fibroin solution shows a pattern with a broad double-tailed asymmetrical distribution (curve b in Fig. 2), ranging from the maximum molecular weight of standard (669 kDa) to below the minimum standard (12.4 kDa). This behavior may be the result of aggregation and hydrolysis/degradation processes of native protein deriving from degumming and/or dissolution in the solvent system. The conformational transition after treatment with ethanol was carried out by means of infrared spectroscopy and DSC analysis. The FTIR spectra for native fibroin and scaffolds are reported in Figure 3: spectrum of silk fibroin considered as a control, shows peaks at 1619, 1514, and 1265 cm - 1; before treatment with ethanol, regenerated silk fibroin scaffolds show absorption bands at 1640, 1530, and 1235 cm - 1, whereas after treatment with ethanol peaks were recorded at 1621, 1510, and 1224 cm - 1. Before ethanol treatment, DSC analysis points out glass transition at 175.76C and exothermic event of crystallization at 210.61C, characteristics of random coil conformation (Fig. 4B). These peaks are absent in silk fibroin (Fig. 4A) and after that scaffolds are treated with ethanol (Fig. 4C) because of the conformational transition of silk fibroin. The morphological investigation of regenerated fibroin scaffold by the analysis of SEM images indicates that silk fibroin regeneration process provides a stable, uniform, smooth surface, which potentially promotes cell adhesion and proliferation (Fig. 5a). The treatment with ethanol induces the b-sheet transition without changing the size distribution of pores (Fig. 5b). Adipose-derived SVF was obtained from intra-articular and subcutaneous adipose tissue as a control: for each subject, the volume of treated tissue, recovered cells, viability, cell yield, and live cell yield are reported in Table 2. The data evaluated with Levene’s test demonstrated the equality of variance. An ANCOVA linear model indicated that BMI and treated tissue volume influence the cell yield (respectively, p = 0.011 and

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FIG. 4. Differential scanning calorimetry patterns of silk fibroin (A), and scaffolds before (B) and after (C) treatment with ethanol.

p = 0.004). BMI and treated tissue volume are in direct and in inverse proportion to cell yield, respectively; this may be due to the higher dimensions of the biopsy in subjects with higher BMI. The bioptical site also influences the cell yield ( p = 0.006). Further, ANCOVA analysis indicates that age and gender do

FIG. 5. SEM images of silk fibroin scaffold: before (a) and after (b) ethanol treatment.

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CHLAPANIDAS ET AL. Table 2. Subcutaneous and Intra-articular Treated Tissue Volume, Recovered Cells, Viability, Cell Yield, and Live Cell Yield, as a Function of Each Subject Recovery layer

Treated tissue volume (mL)

Recovered cells (millions)

Viability (%)

Cell yield (millions/mL tissue)

Live cell yield (millions/mL tissue)

Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular Subcutaneous Intra-articular

15.0 5.0 5.0 4.0 3.0 4.0 2.0 7.0 2.0 10.0 2.0 12.0 5.0 5.0 7.5 4.0 3.0 5.0 3.5 4.0 2.0 4.0 5.0 4.0 3.5 3.0 5.0 4.0 4.0 2.5 4.5 2.5 3.5 2.5 4.0 2.0 1.0 2.5 2.0 2.0 5.0 4.0 4.0 2.5 3.0 4.0 5.0 4.0

3.73 6.15 14.63 14.13 2.44 23.25 188.55 30.39 94.50 27.23 234.60 63.76 186.45 63.56 10.50 2.44 50.42 32.10 8.00 23.30 9.95 5.29 20.96 12.12 19.17 6.84 36.74 12.76 126.17 9.75 38.87 11.39 50.61 15.30 3.42 10.87 2.00 1.49 25.32 3.12 100.00 26.06 20.21 23.63 15.29 7.64 5.30 3.16

86 78 87 89 87 75 82 52 76 67 81 70 67 58 71 85 81 88 77 81 81 73 77 83 87 83 83 84 95 82 87 83 82 88 73 84 70 74 85 81 77 76 75 68 65 69 73 62

0.25 1.23 2.93 3.53 0.81 5.81 94.28 4.34 47.25 2.72 117.30 5.31 37.29 12.71 1.40 0.61 16.81 6.42 2.29 5.83 4.98 1.32 4.19 3.03 5.48 2.28 7.35 3.19 31.54 3.90 8.64 4.55 14.46 6.12 0.86 5.43 2.00 0.60 12.66 1.56 20.00 6.52 5.05 9.45 5.10 1.91 1.06 0.79

0.20 1.00 2.50 3.10 0.70 4.40 77.30 2.30 35.90 1.80 95.00 3.70 25.00 7.40 1.00 0.50 13.60 5.60 1.80 4.70 4.00 1.00 3.20 2.50 4.80 1.90 6.10 2.70 30.00 3.20 7.50 3.80 11.90 5.40 0.60 4.60 1.40 0.40 10.80 1.30 15.40 5.00 3.80 6.40 3.30 1.30 0.80 0.50

Subject 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 10 11 11 12 12 13 13 14 14 15 15 16 16 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24

Table 3. Descriptive Statistics for Cell Yield, Viability, and Live Cell Yield as a Function of Collection Site Collection site

Cell yield (millions/mL tissue)

Viability (%)

Live cell yield (millions/mL tissue)

Intra-articular Subcutaneous

3.72a (1.65–5.83) 5.29b (2.07–19.20)

79.5 (69.3–83.9) 82.0 (73.5–85.8)

2.90a (1.30–4.68) 4.40b (1.50–14.95)

Median and 1st–3rd quartile (in parentheses). Different letters correspond to significant difference ( p < 0.005) between groups (Wilcoxon test).

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FIG. 6. Microphotographs of SVF and chondrocytes cultured on fibroin or hyaluronic acid scaffolds for 7, 10, and 21 days. (A, B) SEM images after 7 days culture with SVF. (C–L) Hematoxylin/eosin staining, original magnification: 40 · . SVF, stromal vascular fraction.

not influence the output variables. A multiple linear regression model indicates that BMI is in inverse proportion to age and direct proportion to treated tissue volume. This could mean that elderly subjects have a BMI higher that the younger and that an increasing of BMI corresponds to greater adipose tissue recovery. The results were analyzed independently from subject-related variables (age, gender, and BMI) using Wilcoxon test (Table 3): the collection site influences cell yield and live cell yield, but not viability. After 1 week of culture, stromal cells adhere in wide clusters, homogeneously coating the fibroin scaffold surface (Fig. 6A, C), whereas on the surface of the hyaluronic acid scaffold some aggregates are visible; presumably, these can be ascribed to the formation of nonstructured extracellular matrix produced by the SVF, basically collagen (Fig. 6B, D). When chondrocytes were cultured on both scaffolds, cells seem to adhere to fibroin to a lesser amount without cluster formation (Fig. 6E, G), with respect to hyaluronic acid (Fig. 6F, H), after 10 and 21 days of culture. After 10 and 21 days of co-culture SVF promotes chondrocytes adherence and proliferation on regenerated silk fibroin scaffold (Fig. 6I, K), whereas ambiguous histological images were obtained from co-cultures on hyaluronic acid (Fig. 6J, L). To identify if adherent cells were stromal cells or pooled stromal cells/ chondrocytes, an immunofluorescence microscopical investigation using both stemness and extracellular matrix markers was performed after 21 days of co-culture. Images indicate that either fibroin or hyaluronic acid present a somewhat strong self-fluorescence, which lead to ambiguous results: notwithstanding, cells appear to be negative to CD34 (Fig. 7A, B) and CD90 (Fig. 7C, D), whereas on fibroin scaffold, cells seem to be embedded in a type II collagen extracellular matrix (Fig. 7E). The same behavior was not observed for co-culture in hyaluronic acid (Fig. 7F).

FTIR spectra for polypeptides and proteins generally show 9 main absorption bands, named amide bands: they are indicated as amide A, B, I, II, III, IV, V, VI, and VII. The most significant for fibroin conformational analysis are amide I, II, and III; the amide I band (1650–1630 cm - 1) is uniquely due to the C=O bond stretching; amide II (1540– 1520 cm - 1) is related to the plane bending of N-H group and the stretching of C-N group. Finally, the amide III (1270–

Discussion Silk fibroin solution was characterized in terms of amino acid composition and mass weight distribution, using gland silk as a control. The obtained values agree with those reported by Vasconcelos et al.17; this aminoacidic pattern results from two distinct fibroin polypeptides, the heavy chain (*390 kDa) and the light chain (*25 kDa). Since the proceeding of silk extraction does not modify the gross aminoacid composition, we can argue that the solubilization technique does not impair the aminoacid stability.

FIG. 7. Immunofluorescence staining: microphotographs of co-cultures on fibroin or hyaluronic acid scaffolds after 21 days. (A, B) CD34/TRITC (red) and DAPI (blue). (C, D) CD90/TRITC (red) and DAPI (blue). (E, F) Type II-collagen/ TRITC (red) and DAPI (blue).

1732 1230 cm - 1) results from the combination of the stretching of C-N bond and the C=O bond vibration.17–19 Moreover, the positioning of the characteristic bands indicates the conformation of protein materials: for amide I a wave number of *1650 cm - 1 is related to a random coil structure, whereas a wave number of *1630 cm - 1 corresponds to a b-sheet structure, and the a-helix conformation finds its peaks at *1658 cm - 1.17,20,21 For amide II, the random coil conformation is at *1540 cm - 1, and the b-sheet structure the wave number corresponds to *1520 cm - 119; for amide III, the random coil shows a peak at *1230 cm - 1 and the b-sheet structure at *1270 cm - 1. The wave number of 1619 cm - 1 in amide I indicates a great b-sheet component in gland fibroin, maintained in the ethanol-treated scaffold (1621 cm - 1); before ethanol treatment, the random coil conformation was more marked (1640 cm - 1). The same conclusions can be drawn for the peaks for amide II; amide III gave ambiguous results, with peak shift toward random coil structure after ethanol treatment. This could be linked to the fact that amide III depends on several intra- and intermolecular interactions: for this reason, the silk secondary structure analysis is often based on the region of amide I selected from the entire spectrum.22 The calorimetry results are coherent with the findings of Um et al.18 and Vasconcelos et al.17 and confirm that an ethanol treatment of regenerated fibroin induces a modification of the cristallinity degree, leading to a predominance of b-sheet structure. The regenerated fibroin treated with ethanol was employed as a scaffold for tissue engineering, and intrapatellar fat pad has been selected to yield SVF as a feeder layer. Although the cell amount recovery was lower in intraarticular tissues with respect to subcutaneous ones, the choice fell on Hoffa’s body SVF since it could be the biological stem cell niche for potential regeneration of intraarticular tissues, and the viability was similar to that of subcutaneous SVF. In the present work, the co-culture of SVF and chondrocytes seems to improve the quality of the in vitro reconstructed tissue in terms of cell density. The hypothesis that IFP could be the articular cartilage niche for mesenchymal stem cells is consistent with Becerra et al.,13 who propose that the self-renewal in avascular tissues depends on neighboring tissues. Therefore, intra-articular SVF was cultured on regenerated silk fibroin scaffolds for 7 days to promote chondrocytes adhesion, as feeder layers. Hyaluronic acid scaffold was employed as a control because it is considered a natural ligand of CD44 receptor on chondrocytes surface.23 Mauney and colleagues24 used bone marrow and adiposederived stem cells to evaluate the cell adhesion and the integrity of 3D silk fibroin scaffold. Results indicate that 3D silk fibroin scaffold is an appropriate material for tissue engineering applications. Meinel and colleagues25 used bone marrow stem cells and evaluated the differentiation of these cells on 3D silk fibroin scaffold. Bone marrow stem cells were isolated, expanded in monolayer, and then seeded on 3D fibroin scaffold in chondrogenic medium containing transforming growth factor b. After 4 weeks, cartilage-like tissue was observed. Uebersax et al.26 promoted the chondrogenic differentiation of bone marrow stem cells also by embedding insulin-like growth factor I in a silk fibroin scaffold; results indicate that insulin-like growth factor-I and transforming

CHLAPANIDAS ET AL. growth factor-b1 promote the chondrogenic differentiation of stem cells. Moreover, silk fibroin-chitosan scaffold leads to stem cell attachment in 2 h of colture, migration, 3D infiltration, and cell–cell interation.27 In 2006, Wang and colleagues seeded chondrocytes on 3D silk fibroin scaffolds, where cells maintained their round shape; increasing cell density, the production of aggrecan, type II collagen, and SOX-9 increased, whereas type X collagen decreased.28 The negativity or positivity to stemness markers in an in vitro reconstructed tissue is fundamental to characterize an advanced therapy product. Sugiyama et al.29 have proposed to use lethally irradiated ADSCs to improve safety of these cells as feeder layer: results showed that the adipose-derived mesenchymal stem cells were able to generate transplantable epithelial cell sheets, similarly to conventional methods, using NIH/3T3 feeder layers. In conclusion, 3D organized silk fibroin is an ideal biopolymer for cartilage tissue engineering, since it leads to cell attachment, scaffold colonization, and physically cell holding in the area that must be repaired; the use of infrapatellar ADSCs is a promising strategy to promote adhesion and proliferation of chondrocytes to the scaffold as an autologous human feeder layer. Acknowledgments This work was supported by Pavia and Milan Universities, Regime Scozzese Rettificato-Giurisdizione Italiana, and PANAGENESI Project 5167 (Regione Lombardia) Eurostars Project E!5227 FIBROSPHERE. The authors thank Dr. Patrizia Vaghi (Centro Grandi Strumenti, Pavia University) for the immunofluorescence investigation. Disclosure Statement No competing financial interests exist. References 1. Heslot, H. Artificial fibrous proteins: a review. Biochimie 80, 19, 1998. 2. Matsumoto, A., Lindsay, A., Abedian, B., and Kaplan, D.L. Silk fibroin solution properties related to assembly and structure. Macromol Biosci 8, 1006, 2008. 3. Wang, Y., Blasioli, D.J., Kim, H.J., Kim, H.S., and Kaplan, D.L. Cartilage tissue engineering with silk scaffolds and human articular chondrocytes. Biomaterials 27, 4434, 2006. 4. Peterson, L., Vasiliadis, H.S., Brittberg, M., and Lindahl A. Autologous chondrocyte implantation: a long-term followup. Am J Sports Med 38, 1117, 2010. 5. Moseley, J.B., Anderson, A.F., Browne, J.E., Mandelbaum, B.R., Micheli, L.J., Fu, F., and Erggelet, C. Long-term durability of autologous chondrocyte implantation. A multicenter, observational study in US patients. Am J Sports Med 38, 238, 2010. 6. Nuernberger, S., Cyran, N., Albrecht, C., Redl, H., Ve´csei, V., and Marlovits, S. The influence of scaffold architecture on chondrocyte distribution and behavior in matrix-associated chondrocyte transplantation grafts. Biomaterials 32, 1032, 2011. 7. Nejadnik, H., Hui, J.H., Pei Feng Choong, E., Tai, B.C., and Hin Lee, E. Autologous bone marrow-derived mesenchymal stem cells versus autologous chondrocyte implantation: an observational cohort study. Am J Sports Med 38, 1110, 2010.

SILK FIBROIN SCAFFOLD FOR TISSUE ENGINEERING 8. Zuk, P.A., Zhu, M., and Mizuno, H. Multilineage cells from human adipose tissue: Implications for cell-based therapies. Tissue Eng 7, 211, 2001. 9. Hofmann, S., Hagenmu¨ller, H., Koch, A.M., Mu¨ller, R., Vunjak-Novakovic, G., Kaplan, D.L., Merkle, H.P., and Meinel, L. Control of in vitro tissue-engineered bone-like structures using human mesenchymal stem cells and porous silk scaffolds. Biomaterials 28, 1152, 2007. 10. Faustini, M., Bucco, M., Chlapanidas, T., Lucconi, G., Marazzi, M., Tosca, M.C., Gaetani, P., Klinger, M., Villani, S., Ferretti, V.V., Vigo, D., and Torre, M.L. Nonexpanded mesenchymal stem cells for regenerative medicine: yield in stromal vascular fraction from adipose tissues. Tissue Eng Part C Methods 16, 1515, 2010. 11. Gaetani, P., Torre, M.L., Klinger, M., Faustini, M., Crovato, F., Bucco, M., Marazzi, M., Chlapanidas, T., Levi, D., Tancioni, F., Vigo, D., and Rodriguez y Baena, R. Adiposederived stem cell therapy for intervertebral disc regeneration: an in vitro reconstructed tissue in alginate capsules. Tissue Eng Part A 14, 1415, 2008. 12. Ghidoni, I., Chlapanidas, T., Bucco, M., Crovato, F., Marazzi, M., Vigo, D., Torre, M.L., and Faustini, M. Alginate cell encapsulation: new advances in reproduction and cartilage regenerative medicine. Cytotechnology 58, 49, 2008. 13. Becerra, J., Santos-Ruiz, L., Andrades, J.A., and Marı´-Beffa, M. The stem cell niche should be a key issue for cell therapy in regenerative medicine. Stem Cell Rev Rep 2010 [Epub ahead of print]; DOI: 10.1007/s12015-010-9195-5. 14. Wickham, M.Q., Erickson, G.R., Gimble, J.M., Vail, T.P., and Guilak, F. Multipotent stromal cells derived from the infrapatellar fat pad of the knee. Clin Orthop 412, 196, 2003. 15. Mathur, A.B., Tonelli, A., Rathke, T., and Hudson, S. The dissolution and characterization of Bombyx mori silk fibroin in calcium nitrate-methanol solution and the regeneration of films. Biopolymers 42, 61, 1997. 16. Zuk, P.A., Zhu, M., Ashjan, P., De Ugarte, D.A., Huang, J.I., Mizuno, H., Alfonso, Z.C., Fraser, J.K., Benhaim, P., and Hedrick, M.H. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 13, 4279, 2002. 17. Vasconcelos, A., Freddi, G., and Cavaco-Paulo, A. Biodegradable materials based on silk fibroin and keratin. Biomacromolecules 9, 1299, 2008. 18. Um, I.C., Kweon, H.Y., Park, Y.H., and Hudson, S. Structural characteristics and properties of the regenerated silk fibroin prepared from formic acid. Int J Biol Macromol 29, 91, 2001. 19. Ha, S.W., Tonelli, A.E., and Hudson, S.M. Structural studies of Bombyx mori silk fibroin during regeneration from solution and wet fiber spinning. Biomacromolecules 6, 1722, 2005. 20. Hu, X., Kaplan, D., and Cebe, P. Determining beta-sheet crystallinity in fibrous proteins by thermal analysis and infrared spectroscopy. Macromolecules 39, 6161, 2006.

1733 21. Chen, X., Shao, Z., Marinkovic, N.S., Miller, L.M., Zhou, P., and Chance, M.R. Conformation transition kinetics of regenerated Bombyx mori silk fibroin membrane monitored by time-resolved FTIR spectroscopy. Biophys Chem 89, 25, 2001. 22. Lawrence, B.D., Omenetto, F., Chui, K., and Kaplan, D.L. Processing methods to control silk fibroin film biomaterial features. J Mater Sci 43, 6967, 2008. 23. Chow, G., Knudson, C.B., Homandberg, G., and Knudson, W. Increased expression of CD44 in bovine articular chondrocytes by catabolic cellular mediators. J Biol Chem 270, 27734, 1995. 24. Mauney, J.R., Nguyen, T., Gillen, K., Kirker-Head, C., Gimble, J.M., and Kaplan, D.L. Engineering adipose-like tissue in vitro and in vivo utilizing human bone marrow and adipose-derived mesenchymal stem cells with silk fibroin 3D scaffolds. Biomaterials 28, 5280, 2007. 25. Meinel, L., Hofmann, S., Karageorgiou, V., Zichner, L., Langer, R., Kaplan, D.L., and Vunjak-Novakovic, G. Engineering cartilage-like tissue using human mesenchymal stem cells and silk protein scaffolds. Biotech Bioeng 88, 379, 2004. 26. Uebersax, L., Merkle, H.P., and Meinel, L. Insulin-like growth factor I releasing silk fibroin scaffolds induce chondrogenic differentiation of human mesenchymal stem cells. J Control Release 127, 12, 2008. 27. Altman, A.M., Gupta, V., Rı´os, C.N., Alt, E.U., and Mathur, A.B. Adhesion, migration, and mechanics of human adipose tissue derived stem cells on silk fibroin-chitosan matrix. Acta Biomater 6, 1388, 2010. 28. Wang, Y., Kim, H.J., Vunjak-Novakovic, G., and Kaplan, D.L. Stem cell-based tissue engineering with silk biomaterials. Biomaterials 27, 6064, 2006. 29. Sugiyama, H., Maeda, K., Yamato, M., Hayashi, R., Soma, T., Hayashida, Y., Yang, J., Shirakabe, M., Matsuyama, A., Kikuchi, A., Sawa, Y., Okano, T., Tano, Y., and Nishida, K. Human adipose tissue-derived mesenchymal stem cells as a novel feeder layer for epithelial cells. J Tissue Eng Regen Med 2, 445, 2008.

Address correspondence to: Massimo Faustini, Ph.D. Dipartimento di Scienze e Tecnologie Veterinarie per la Sicurezza Alimentare Universita` di Milano Via Celoria 10 Milan 20133 Italia E-mail: [email protected] Received: November 3, 2010 Accepted: February 18, 2011 Online Publication Date: June 8, 2011

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