Chemically Functionalized Silk for Human Bone Marrow-Derived

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Chemically Functionalized Silk for Human Bone Marrowderived Mesenchymal Stem Cells Proliferation and Differentiation Ke Zheng, Ying Chen, Wenwen Huang, Yinan Lin, David L Kaplan, and Yimin Fan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03518 • Publication Date (Web): 13 May 2016 Downloaded from http://pubs.acs.org on May 17, 2016

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ACS Applied Materials & Interfaces

Chemically Functionalized Silk for Human Bone Marrow-Derived Mesenchymal Stem Cells Proliferation and Differentiation Ke Zheng1,2, Ying Chen2, Wenwen Huang2, Yinan Lin2, David L. Kaplan*,2 and Yimin Fan*,1

1 Jiangsu Key Lab of Biomass-based Green Fuel & Chemicals, College of Chemical Engineering, Nanjing Forestry University, Nanjing 210037, China

2 Department of Biomedical Engineering, 4 Colby Street, Tufts University, Medford, MA 02155, United States

AUTHOR INFORMATION

Corresponding author: Prof. Yimin Fan: [email protected]; Prof. David L. Kaplan: [email protected]

Keywords: silk fibroin; oxidation; carboxyl groups; serine; mesenchymal stem cells

Abstract: To produce biocompatible, mechanically robust and conductive materials for bone tissue engineering, chemical oxidation using sodium hyprochlorite (NaClO) was

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utilized to introduce carboxyl groups onto silk fibroin (SF). A final carboxyl content of 1.09 mM/g SF was obtained, corresponding to ~47% of the primary hydroxymethyl groups on the silk. Interestingly, both infrared spectroscopy and circular dichroism spectra demonstrated that the resulting oxidized silk (OxSF) self-assembled into β-sheet structures in aqueous conditions and this contributed to the mechanical properties of the as-prepared silk-based scaffolds and the mineralized OxSF scaffolds (M-OxSF). The OxSF scaffolds had a compressive modulus of 211±75 KPa in the hydrated state, 10 times higher than that of the SF scaffolds, and the modulus of the M-OxSF scaffolds was increased to 758±189 KPa. Human bone marrow-derived mesenchymal stem cells (hMSCs) grown on the scaffolds during osteogenesis showed that the OxSF scaffolds supported the proliferation and differentiation of hMSCs in vitro.

1. INTRODUCTION Biomaterials derived from Bombyx mori silk fibroin (SF) represent promising candidates for bone tissue regeneration due to their mechanical properties, processing plasticity, and low inflammatory and immunogenic responses1-2. For bone grafting as well as guided neotissue formation, successfully engineered materials must be (i) highly porous, interconnected microstructures to promote cell migration and proliferation3-6; (ii) provide an environment that is conducive to cell differentiation and the deposition of new tissue7-12; (iii) provide sufficient mechanical properties to meet

minimal

mechanical

requirements13-18,

(iv)

surface


roughness,

and

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osteoconductivity1, 15, 19-20. Toward these goals, a variety of chemical, structural and biomechanical modifications of SF have been studied to improve functionality for bone tissue engineering21-25. These studies mainly focused on improving osteoconductivity26-31, modifying SF side chains to immobilize growth factors to facilitate bone formation7-8, 32-33, and reinforcement to match mechanical needs20, 34-36. These studies demonstrated that SF protein-based materials can be processed to various forms to support bone tissue engineering. Indeed, all of these works were remarkable, but still need cumbersome processing to meet the requirements of bone tissue engineering. Therefore, simplified procedures would be useful toward the needs of bone formation.

Here, a one-step oxidation method was used to introduce carboxyl groups onto the SF, providing a surface favorable for hydroxyapatite (HA) mineralization as well as bone matrix formation15, 37. The abundance of the polar and negatively charged carboxyl group can also alter the assembly process of the protein38-39, and provide chemical handles for further modifications or composites as needed1,

24

. Meanwhile, the

oxidation of SF improved the functionality and mechanical properties of the as-prepared materials simultaneously.

The objective of this study was to optimize the oxidation of SF to functionalize the protein and determine the influence of this modification on both the structural assembly of the SF as well as the role of this modification in mineralization in vitro by human bone marrow-derived mesenchymal stem cells (hMSCs). The oxidized silk



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fibroin (OxSF) was also processed to scaffolds by freeze-drying40, and then mineralized in a calcium chloride-disodium hydrogen phosphate solution (Ca:P was 10:6).

2. EXPERIMENTAL SECTION

2.1 Preparation of Silk Fibroin Solution

Aqueous silk fibroin solution was prepared from Bombyx mori silkworm cocoons supplied by Tajima Shoji Co. according to our previously published procedures17. Cocoons were cut to pieces and boiled for 30 min in an aqueous solution of 0.02 M sodium carbonate, followed by thorough washing in deionized water and air drying. The purified silk fibroin was then dissolved in 9.3 M LiBr solution at 60˚C for 4 h, yielding a 20 wt% solution. This solution was filtered through a 5 µm syringe filter, and then dialyzed (Pierce, molecular weight cut off 3,500) against distilled water for 3 days with changing of the water for several times. The final concentration of aqueous silk fibroin solution was about 8 wt%, determined by weighing the remaining solid after drying.

2.2 Oxidation of Silk Fibroin Solution The oxidation of silk fibroin solution was initiated by adding a desired amount of sodium hypochlorite (NaClO) solution (0-5 mmol of NaClO per gram of protein). The pH of the mixture was maintained at 10 at room temperature by continuous addition of 0.5 M NaOH using a pH-Stat titration system. When no consumption of the alkali


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was observed, the reaction was quenched by adding drops of 0.5 M hydrochloric acid (HCl) to adjust the pH to 7. After oxidation, the solution was dialyzed (Pierce, molecular weight cut off 10,000) against distilled water at 4˚C for 2 days to purify the oxidized silk solution, followed by weighing the remaining protein after drying.

2.3 Determination of Carboxyl Content The carboxyl content of the oxidized silk solution was determined by the electrical conductivity titration method41. A 2.5 mL aliquot of silk solution (0.1g of protein) was added to water (60 mL) and stirred for 5 min. Then 0.1 M HCl was added to the mixture to set the pH in the range of 2.5-3.0. A 0.05 M NaOH solution was added at a rate of 0.1 mL/min up to pH 11 using a pH-Stat titration system. The carboxylate content was calculated using the conductivity and pH curves.

2.4 Secondary Structure of Oxidized Silk Fibroin

Circular Dichroism (CD) spectra of oxidized solution were obtained on an AVIV Model 420 CD spectrometer (AVIV Biomedical, Lakewood, NJ, USA) using glass cuvettes with a 1 mm path length. SF solutions were equilibrated for 24 h at 4˚C and diluted to 0.2 mg/mL for measurement. Wavelength scans were collected from 190 to 260 nm in 0.5 nm steps with a 4 s averaging time, 1.0 nm band width and repeated three times. The SF and OxSF solutions were air-dried at room temperature to obtain films with a thickness of about 0.1 mm. The secondary structure of the films was analyzed by Fourier-Transform Infrared (FTIR) on JASCO FTIR 6200 spectrometer (JASCO, Tokyo, Japan) equipped with an attenuated total reflection (ATR) detector.



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For each measurement, 128 scans were coded with resolution 4 cm-1, with the wave numbers ranging from 600 to 4000 cm-1. A nitrogen atmosphere was used to avoid interference with water absorption.

2.5 Differential Scanning Calorimetry and Thermogravimetric Analysis

Temperature Modulated Differential Scanning Calorimetry (TMDSC) measurements were carried out using a TA Instruments Q100 DSC (TA Instruments, New Castle, DE, USA) equipped with a refrigerated cooling system to obtain total heat flow and reversing heat capacity (rev. Cp). Indium was employed for the heat flow and temperature calibration before sample measurements. The experiments were performed at a heating rate 2˚C/min with a modulation period of 60 s and temperature amplitude of 0.318˚C. A nitrogen purge gas was used at a flow rate of 50 mL/min. The sample mass was about 2-3 mg. Endotherms are presented with downward deflection in heat flow vs. temperature scans. Thermogravimetry (TG) was performed on a TA Instruments Q500 (TA Instruments, New Castle, DE, USA) thermogravimetric analyzer at a heating rate of 2˚C/min from room temperature to 600˚C in a nitrogen purge gas flow of 50 mL/min. The initial sample mass was in the range between 4.0 and 5.0 mg.

2.6 Preparation of Scaffolds for Cell Culture The silk scaffolds were fabricated by freeze-drying25. Briefly, 4 mL of SF or OxSF solution (4 wt%) was adding into disk-shaped containers. Then the containers were placed into -20˚C freezer for 24 h. The ice/silk composite was then lyophilized


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leaving porous scaffolds. To make the silk insoluble in water, the scaffolds were then treated by water annealing42 at 50˚C for 12 h. For further studies, the scaffolds were immersed into 0.1 M calcium chloride (CaCl2) solution for at least 1 hour, then 0.1 M disodium hydrogen pyrophosphate (Na2HPO4) solution was added into the mixture for mineralization (The mole ratio of Ca:P was 10:6). The scaffolds were incubated with the mixture at room temperature for 1 day and then washed by distilled water.

2.7 Pore Size and Porosity Measurement of Scaffolds

The pore size of silk scaffolds was analyzed with ImageJ software (1.48) developed at the US National Institutes of Health. The porosity of the scaffolds was measured by liquid displacement with hexane, as previous reported13. The silk scaffolds were lyophilized and then immersed in a known volume (V1) of hexane in a graduated cylinder for 5 min. The total volume of hexane and the hexane-impregnated scaffold was recorded as V2. The hexane-impregnated scaffold was then removed from the cylinder and the residual hexane volume was recorded as V3. The porosity of the scaffold was calculated and expressed as the following formula.

Porosity (%) = (V1-V3)/(V2-V3)×100%

2.8 Mechanical Properties of Scaffolds

Unconfined compressive mechanical testing of the scaffolds was performed on an Instron 3366 testing frame (Instron Corp., USA) equipped with a 0.1 kN load cell. Tests for all scaffolds were carried out in 0.1 M phosphate buffer saline (PBS) bath



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(BioPuls, Instron Corp., USA) at 37˚C under hydrated conditions. Separately, silk scaffold discs with dimensions of 4 mm diameter and 5 mm height were studied in compressive tests. All tests were with a conventional open-sided (nonconfined) configuration and were performed using a displacement control mode at a rate of 5 mm/min following ASTM standard D1621-04a (standard test method for compressive properties of rigid cellular plastics). After the compression tests, the compressive stress and strain were graphed based on the measured cross-sectional area and sample height (nominal ~4–5 mm, measured automatically at 0.02 N tare load), respectively. The elastic modulus was calculated based on a linear regression fitting of the small strain section that preceded an identifiable plateau region.

2.9 Human Mesenchymal Stem Cell Culture hMSCs were isolated as previously described16. Cells were maintained in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, 1% non-essential amino acids, and 1 ng/mL basic fibroblast growth factor (bFGF) and were plated in 175 cm2 tissue culture flasks at a density of 5×104 cells/cm2. Cells at passage 4 (P4) were used for the experiments. hMSCs (5×105 cells/scaffold) were seeded onto three-dimensional scaffolds (length×width×height; 3 mm×3 mm×3 mm) and cultured in a humidified incubator at 37˚C and 5% CO2. After 3 days, the medium was removed and cultures were maintained in individual wells of 12-well plates. The medium was changed every other day. Osteogenic medium consisted of DMEM supplemented with 10%


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FBS, 0.1 mm nonessential amino acids, 50 µg/mL ascorbic acid-2-phosphate, 10 nm dexamethasone, and 10 mm β-glycerol phosphate in the presence of 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL fungizone. Cultures were maintained at 37°C in a humidified incubator supplemented with 5% CO2. Half of the medium was changed every 2–3 days. The scaffolds were cultured for 28 days and samples removed for analysis at day 3 and weeks 1, 2, 3 and 4.

2.10 Examination of Human Mesenchymal Stem Cell Viability

The viability of the hMSCs on the scaffolds was monitored by live/dead stain (Invitrogen, USA) following the manufacturers’ protocol. Briefly, scaffolds seeded with cells were washed with PBS, incubated in 2 mM calcein AM (staining live cells) in medium for 1 hour at 37˚C. The scaffolds were scanned using a Keyence BZ-X700 digital microscope (Keyence Canada Inc., Mississauga) with Z-series capability. The scaffolds were observed under the microscope with a filter set for DAPI (Ex/Em: 350/470nm), and GFP/FITC (Ex/Em: 488/514 nm). Three dimensional maximum projection images were assembled with BZ-II Image Analysis Application (Keyence Canada Inc., Mississauga).

2.11 Gene Expression using Real-Time Polymerase Chain Reaction

RNA isolation and real-time reverse transcription polymerase chain reactions (RT-PCR) were carried out using following protocols. Total RNA was extracted from cells using a Qiagen Mini mRNA Extraction kit (Qiagen, Valencia, CA, USA) following the supplier’s instructions. Briefly, scaffolds (n=3) were harvested and



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washed in PBS, transferred into 2 mL Eppendorf tubes and kept in RNAlater solution at -20°C. After thawing, the tubes were centrifuged at 12,000g for 10 min to pellet the cells and scaffolds. The supernatants were removed and the scaffolds were cut into small pieces before being lysed in 600 µL buffer RLT (Qiagen, Valencia, CA) with 1% β-mercaptoethanol (β-ME). The supernatants of the lysates after centrifugation were collected and homogenized with a QIA shredder spin column (Qiagen, Valencia, CA). RNA was reverse-transcribed using a High-Capacity cDNA Reverse Transcription Kit (Invitrogen, USA) following the manufacturer’s instructions. Six nanograms of cDNA were used for real-time PCR amplification for each well, using primer sequences shown in Table S1. Gene expression profiles were obtained for alkaline phosphatase (ALP), collagen Type 1 (Col1a1), osteopontin (OPN), and osteocalcin (OC). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene. For each gene tested three experimental replicates and three biological replicates were run. Gene expression levels were normalized to the GAPDH mRNA level.

2.12 Statistical Analysis

Data are presented as mean ± standard deviation (n= 5). A two tailed t-test was performed to compare means between two groups, and analysis of variance (ANOVA) was performed to compare means of multiple groups. P-values ≤ 0.05 were considered significant.

3. Results and Discussion


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3.1 Sodium Hypochlorite Oxidation of Silk Fibroin

The composition of SF is mainly glycine-alanine-glycine-alanine-glycine-serine (GAGAGS) repeats which self-assemble into an anti-parallel β-sheet structure by hydrogen bonding and hydrophobic interactions. These β-sheet structures leading to crystallinity of silk proteins, and controlling the mechanical properties of materials13. As non-reactive amino acids, glycine and alanine, few options for functionalization are present. Most previous studies focused on the modification of the active residues in silk side chains (e.g., limited acid/amino groups and extensive tyrosine hydroxyl groups) with known chemistries24-25, 43. Reactions between NaClO and proteins have been widely studied, while only previously reported for the degradation or dissolution of SF44. Figure 1A presents the oxidation reaction between NaClO and SF where the hydroxymethyl groups on the serine residues were oxidized to carboxyl groups by NaClO. Stoichiometrically, to oxidize 1 mol of the primary hydroxymethyl group to a carboxyl group, 2 mol of NaClO are required41, 45. The serine accounts for about 12.1% relatively to the total amino of SF46-47, which means an additional 1.62 mmol of carboxyl groups can be obtained in SF as long as all serine residues are oxidized (see supporting information). The extensive tyrosine hydroxyl groups (~ 5% of the amino acids per SF chain) were excluded here because the main reaction with NaClO and tyrosine is chlorination with the aromatic ring48. Otherwise, there are several amino groups in the SF protein that may be attacked by NaClO as well, but the contribution here is negligible due to the minor abundance47-48.



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The pure SF solution had an initial carboxylate concentration of 0.335 mmol/g of protein (Figure 1B) with respect to the aspartic and glutamic acids47. Thereafter, the different carboxyl contents of silk solution were plotted in Figure 1B with respect to the amount of NaClO added to the reaction. When NaClO addition was below 2 mM/g SF, the carboxyl content increased almost linearly. The final carboxyl content 1.09 mM/g was achieved when NaClO was added to 2 mM/g SF, which roughly corresponded to 47% of the primary hydroxymethyl groups on the original SF had been oxidized. After oxidation, the carboxylic silk solution was dialyzed against distilled water for 3 days. Figure 1C shows the relationship between the amount of NaClO added and the weight remaining of OxSF protein after dialysis. When NaClO was added below 2 mM/g, more than 87% of the silk protein can be obtained with respect to the initial SF, suggesting limited degradation during the reaction. However, a significant weight loss, 72% of silk protein remained, was found when 5 mM/g NaClO was added. These results suggested that excess amounts of NaClO maybe degrade the silk.

3.2 The Effect of Oxidation on Structure

In Figure 2A, CD measurements depict the secondary structure of OxSF solutions with NaClO concentrations from 0.2 to 2 mM/g. The 0.2 mM NaClO oxidized silk solution was similar to the control SF solution with a negative peak at 203 nm corresponding to random coil structure49-50. The increased NaClO content from 0.5-2 mM/g SF, resulted in a shift of the negative peak of the OxSF solution to 218 nm and a


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clear maximum at 208 nm, indicative of the β-sheet structure49, 51. The intensity of the CD signal at 218 nm increased as a function of NaClO concentration, thus the amount of β-sheet was enriched.

The corresponding FTIR spectra (Figure 2B) shows the amide I peaks at 1700–1600 cm-1, known vibrations to provide information on the secondary structures of protein backbones. The SF and 0.2 mM/g NaClO oxidized OxSF films showed absorption peaks at 1655 cm-1, which were assigned to random coil and/or helical conformations52. The spectrum of OxSF oxidized by NaClO concentrations higher than 0.5 mM showed absorption peaks at 1700 cm-1 and 1630 cm-1. These peaks at 1630 cm-1 were attributed to β-sheet, and the shoulder peak at 1700 cm-1 was assigned to β-turn conformations associated with the antiparallel β-sheet structure52. However, a carboxyl vibration peak at 1700 cm-1 was dominated by peaks corresponding to the protein backbone.

It is interestingly to note that both the CD and FTIR measurements indicated the presence of β-sheet structure in the as-prepared OxSF solution. Silk aqueous solution spontaneously assembles into a β-sheet structures by intermolecular crosslinks24, 53, while this assembly can take weeks to occur but can induced by lowering the pH, increasing salt concentration or the addition of organic solvents53. In the present case, the self-assembly of the OxSF solution occurred in neutral aqueous solution at room temperature in 2 days. This change in the kinetics of β-sheet formation was likely due to the carboxylation of the protein, which altered charge density as well as hydrogen



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bonding39, 54. Previous research showed that crystallization of artificial polypeptides consisting of alanylglycine sequences interspersed with glutamic acid residues produced β-sheet structures in the solid state39. Moreover, the ionization of carboxyl groups influence protein structure and resulted in stronger interactions with the solvent or with molecular chains in the resultant OxSF55. During the crystal growth process of the silk protein, these interactions should segregate the carboxyl residues to the surface of the growing aggregate as well as induce lamellar assembly39. However, the OxSF with 0.2 mM/g SF NaClO did not show significant β-sheet structure in the CD spectra, which was attributed to the lower content of carboxyl groups.

TMDSC was used to further study the glass transition and nonisothermal crystallization of SF and OxSF. Figure 3A illustrates the total heat flow as a function of temperature of the SF and OxSF samples oxidized with 2 mM/g of NaClO. Both SF and OxSF showed an endotherm at 100˚C due to the evaporation of bound water. The heat flow of SF showed a step change at the glass transition temperature near 178˚C, while this transition moved to a higher temperature in the OxSF sample. During the scan, there was a large exothermic peak at 227˚C in the SF sample, which was attributed to the nonisothermal crystallization of silk fibroin, which was weakened in the OxSF sample, suggesting that the OxSF sample at least partially crystalized during the oxidation. After the appearance of the crystallization peak, the total heat flow of both samples decreased at around 250˚C due to onset of thermal degradation. Figure 3B showed the specific reversing heat capacity of the glass transition for SF and OxSF. With the β-sheet crystallization, the glass transition temperature (Tg) of OxSF shifted


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to 186˚C. The β-sheets in the crystalline regions performed a similar role as physical cross-links in SF, causing an increase of Tg and a broadening of the relaxation process at Tg, thus, consistent with the CD and FTIR results. The TG curve (Figure 3C) showed that the OxSF was similar to the SF in weight loss at the elevated temperatures. The water content in each sample was about 6%. The second step of weight loss for SF and OxSF started at about 210 and 200˚C, respectively. The OxSF samples had a lower degradation temperature beginning at 200˚C, however, residue of the OxSF samples were around 40% after 550˚C, higher than for the control samples. This difference may also be due to some residual salt in t the carboxyl group containing samples.

3.3 Mechanical Properties of Oxidized Silk Scaffolds

To further compare the properties of OxSF to SF, OxSF and SF solutions were formed into sponge scaffolds using freeze-drying and water annealing42 (Figure 4A). The SF solution with 4% concentration was used to obtain scaffolds with pore sizes around 250 µm, as the preferred pore size for bone regeneration are generally in the 100–350 mm range13, 40. The resulting free-standing scaffolds formed highly interconnected and porous structures (Figure 4B). The porosity of SF and OxSF scaffolds were 98.9±0.2 and 98.7±0.1%, respectively. The pore size of OxSF scaffolds was 180±25 µm, which is similar to the silk scaffolds (200±40 µm), and consistent with previous results1, 29. The oxidation of SF enhanced the compressive modulus of the scaffolds (Figure 4C). The OxSF scaffolds had a compressive modulus of 211±75 KPa in the



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hydrated state which was at least 10 times higher than that of the SF scaffolds. These higher mechanical properties for the OxSF scaffolds was due to the by higher β-sheet content, based on the stronger absorption at 1630 cm-1 of OxSF compared with the SF in FTIR spectra (Figure 2B).

Mineralization of OxSF and SF scaffolds was carried out to induce and support the formation of mineralized bone matrix with hMSCs. The scaffolds were incubated in a CaCl2-Na2HPO4 solution (0.1 mM, Ca:P molar ratio of 10:6) at room temperature for 24h. As expected, the higher carboxyl group content of OxSF induced the nucleation of hydroxyapatite. The OxSF scaffolds showed stronger mineralization than the SF scaffolds (red arrows in Figure 4D,E). SEM analysis and TG curves (Figure S1) confirmed that higher content of minerals were obtained on M-OxSF scaffold. The OxSF scaffolds were covered with a thick layer of minerals, while only sparse mineralization was observed on the SF scaffolds. The SEM images also confirmed that the pores and structure of the mineralized scaffolds were similar to the initial scaffold features. The XPS and XRD confirmed that the crystals were hydroxyapatite (Figures S4, S5). Mechanical analysis revealed that the scaffolds were further reinforced by the mineralization15. Due to the higher density of mineral in OxSF scaffolds, the modulus of the scaffolds increased from 211±75 to 758±189 KPa, close to the values of some scaffolds reinforced by silk fibers or particles16-17. In comparison, the SF scaffolds showed much lower values increased after mineralization, from 18±6 to 21±8 KPa. These mineralized-OxSF (M-OxSF) and mineralized-SF (M-SF) scaffolds could be considered as biodegradable support


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conduits for native cells to proliferate and differentiate.

3.4 Cell viability and Osteogenic Differentiation of hMSCs within SF and OxSF Scaffolds

To investigate the effect of the oxidation of the silk scaffolds on hMSC survival and osteogenic differentiation, hMSCs were seeded on SF, OxSF, M-SF, and M-OxSF scaffolds and grown in culture for up to 4 weeks. Cell metabolic activity was quantified using the alamarBlue® assay (Figure S6) and the viability was visualized by live/dead staining each week, while the osteogenic differentiation of hMSCs was evaluated by qRT-PCR using well-defined osteogenic markers (ALP, Col1a1, OPN, OC)10, 28, 56. Live/dead staining at day 3 post cell seeding revealed that the hMSCs were successfully delivered into the porous scaffold bulk space in all groups (data not shown). High cell viability was observed on all 3D scaffold samples, while a significant increase in cell viability was only found in the oxidized silk scaffolds (Figure 5, A-L). hMSC gene expression profiles (Figure 5, M-P) obtained from qRT-PCR indicated that hMSCs cultured in the oxidized scaffolds (OxSF and M-OxSF) showed significantly higher gene expression levels (*p

Chemically Functionalized Silk for Human Bone Marrow-Derived

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