Repair of an articular cartilage defect using adipose-derived stem cells loaded on a polyelectrolyte complex scaffold based on poly(l-glutamic acid) and chitosan

As a synthetic polypeptide water-soluble poly(l-glutamic acid) (PLGA) was designed to fabricate scaffolds for cartilage tissue engineering. Chitosan (CHI) has been employed as a physical cross-linking component in the construction of scaffolds. PLGA/CHI scaffolds act as sponges with a swelling ratio of 760 ± 45% (mass%), showing promising biocompatibility and biodegradation. Autologous adipose-derived stem cells (ASCs) were expanded and seeded on PLGA/CHI scaffolds, ASC/scaffold constructs were then subjected to chondrogenic induction in vitro for 2 weeks. The results showed that PLGA/CHI scaffolds could effectively support ASC adherence, proliferation and chondrogenic differentiation. The ASCs/scaffold constructs were then transplanted to repair full thickness articular cartilage defects (4 mm in diameter, to the depth of subchondral bone) created in rabbit femur trochlea. Histological observations found that articular defects were covered with newly formed cartilage 6 weeks post-implantation. After 12 weeks the regenerated cartilage had integrated well with the surrounding native cartilage and subchondral bone. Toluidine blue and immunohistochemical staining confirmed similar accumulation of glycosaminoglycans and type II collagen in engineered cartilage as in native cartilage 12 weeks post-implantation. The result was further supported by quantitative analysis of extracellular matrix deposition. The compressive modulus of the engineered cartilage increased significantly from 30% of that of normal cartilage at 6 weeks to 83% at 12 weeks. Cyto-nanoindentation also showed analogous biomechanical behavior of the engineered cartilage to that of native cartilage. The results of the present study thus demonstrate the potentiality of PLGA/CHI scaffolds in cartilage tissue engineering.     

Chitosan/polyester-based scaffolds for cartilage tissue engineering: Assessment of extracellular matrix formation

Naturally derived polymers have been extensively used in scaffold production for cartilage tissue engineering. The present work aims to evaluate and characterize extracellular matrix (ECM) formation in two types of chitosan-based scaffolds, using bovine articular chondrocytes (BACs). The influence of these scaffolds’ porosity, as well as pore size and geometry, on the formation of cartilagineous tissue was studied. The effect of stirred conditions on ECM formation was also assessed. Chitosan-poly(butylene succinate) (CPBS) scaffolds were produced by compression moulding and salt leaching, using a blend of 50% of each material. Different porosities and pore size structures were obtained. BACs were seeded onto CPBS scaffolds using spinner flasks. Constructs were then transferred to the incubator, where half were cultured under stirred conditions, and the other half under static conditions for 4 weeks. Constructs were characterized by scanning electron microscopy, histology procedures, immunolocalization of collagen type I and collagen type II, and dimethylmethylene blue assay for glycosaminoglycan (GAG) quantification. Both materials showed good affinity for cell attachment. Cells colonized the entire scaffolds and were able to produce ECM. Large pores with random geometry improved proteoglycans and collagen type II production. However, that structure has the opposite effect on GAG production. Stirred culture conditions indicate enhancement of GAG production in both types of scaffold.     

The effect of type II collagen coating of chitosan fibrous scaffolds on mesenchymal stem cell adhesion and chondrogenesis

The biocompatibility of chitosan and its similarity to glycosaminoglycans (GAG) make it attractive for cartilage tissue engineering. We have previously reported improved chondrogenesis but limited cell adhesion on chitosan scaffolds. Our objectives were to produce chitosan scaffolds coated with different densities of type II collagen and to evaluate the effect of this coating on mesenchymal stem cell (MSC) adhesion and chondrogenesis.Chitosan fibrous scaffolds were obtained by a wet spinning method and coated with type II collagen at two different densities. A polyglycolic acid mesh served as a reference group. The scaffolds were characterized by Fourier-transform infrared spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and type II collagen content. Constructs were analyzed after MSCs seeding via live/dead assay, weight and DNA evaluations, SEM, and TEM. Constructs were cultured in chondrogenic medium for 21 days prior to quantitative analysis (weight, DNA, and GAG), SEM, TEM, histology, immunohistochemistry, and quantitative real time polymerase chain reaction. The cell attachment and distribution after seeding correlated with the density of type II collagen. The cell number, the matrix production, and the expression of genes specific for chondrogenesis were improved after culture in collagen coated chitosan constructs.These findings encourage the use of type II collagen for coating chitosan scaffolds to improve MSCs adhesion and chondrogenesis, and confirm the importance of biomimetic scaffolds for tissue engineering.     

Chitosan scaffolds: interconnective pore size and cartilage engineering

This study was designed to determine the effect of interconnective pore size on chondrocyte proliferation and function within chitosan sponges, and compare the potential of chitosan and polyglycolic acid (PGA) matrices for chondrogenesis. Six million porcine chondrocytes were seeded on each of 52 prewetted scaffolds consisting of chitosan sponges with (1) pores 10 microm in diameter (n=10, where n is the number of samples); (2) pores measuring 10-50 microm in diameter (n=10); and (3) pores measuring 70-120 microm in diameter (n=10), versus (4) polyglycolic acid mesh (n=22), as a positive control. Constructs were cultured for 28 days in a rotating bioreactor prior to scanning electron microscopy (SEM), histology, and determination of their water, DNA, glycosaminoglycan (GAG) and collagen II contents. Parametric data was compared (p=0.05) with an ANOVA and Tukey's Studentized range test. PGA constructs consisted essentially of a matrix containing more cells than normal cartilage. Whereas very few remnants of PGA remained, chitosan scaffolds appeared intact. DNA and GAG concentrations were greater in PGA scaffolds than in any of the chitosan groups. However, chitosan sponges with the largest pores contained more chondrocytes, collagen II and GAG than the matrix with the smallest pores. Constructs produced with PGA contained less water and more GAG than all chitosan groups. Chondrocyte proliferation and metabolic activity improved with increasing interconnective pore size of chitosan matrices. In vitro chondrogenesis is possible with chitosan but the composition of constructs produced on PGA more closely approaches that of natural cartilage.     

Chemical modification of collagen improves glycosaminoglycan retention of their co-precipitates

Being prevalent extracellular matrix components, collagen and glycosaminoglycan (GAG) are co-precipitated as scaffolds for tissue regeneration. However, the amount of GAG incorporated and its long-term retention present a persistent problem. In this study, chemical modifications, namely deamination, methylation and amination, were used to alter the net charge of collagen prior to fabrication of collagen–GAG co-precipitate. While most GAGs were lost in the untreated group and the deaminated group within 1 day, methylation and amination of collagen retained over 20% and 40% GAG after 6 days, respectively. Moreover, over 60% of GAG retention was achieved in the aminated group after cell seeding for 8 days. Furthermore, amination of collagen increased the GAG/hydroxyproline ratio in the co-precipitate to >4.5, approaching that of native nucleus pulposus. Ultrastructural analysis showed that the aminated group contains abundant granular substances resembling the extracellular matrix of native nucleus pulposus. Despite lower initial cell adhesion than untreated, all modified scaffolds promoted proliferation of human mesenchymal stem cells (hMSCs) and showed >95% cell viability at all time points. Cell morphology was distinct among the different groups, being round in the untreated control and methylated groups but elongated in deaminated and aminated groups. hMSCs adhered to scaffolds via collagen receptor integrin α2β1 in all groups, while all but the aminated group showed extensive expression of the general matrix receptor integrin αv. This work reports an effective method, namely amination of collagen, to improve GAG incorporation and retention in collagen–GAG co-precipitates, facilitating the fabrication of GAG-rich collagenous scaffold for intervertebral disc tissue engineering.     

Chondrogenic differentiation of rat MSCs on porous scaffolds of silk fibroin/chitosan blends

Adult bone marrow derived mesenchymal stem cells are undifferentiated, multipotential cells and have the potential to differentiate into multiple lineages like bone, cartilage or fat. In this study, polyelectrolyte complex silk fibroin/chitosan blended porous scaffolds were fabricated and examined for its ability to support in vitro chondrogenesis of mesenchymal stem cells. Silk fibroin matrices provide suitable substrate for cell attachment and proliferation while chitosan are promising biomaterial for cartilage repair due to it’s structurally resemblance with glycosaminoglycans. We compared the formation of cartilaginous tissue in the silk fibroin/chitosan blended scaffolds with rat mesenchymal stem cells and cultured in vitro for 3 weeks. Additionally, pure silk fibroin scaffolds of non-mulberry silkworm, Antheraea mylitta and mulberry silkworm, Bombyx mori were also utilized for comparative studies. The constructs were analyzed for cell attachment, proliferation, differentiation, histological and immunohistochemical evaluations. Silk fibroin/chitosan blended scaffolds supported the cell attachment and proliferation as indicated by SEM observation, Confocal microscopy and metabolic activities. Alcian Blue and Safranin O histochemistry and expression of collagen II indicated the maintenance of chondrogenic phenotype in the constructs after 3 weeks of culture. Glycosaminoglycans and collagen accumulated in all the scaffolds and was highest in silk fibroin/chitosan blended scaffolds and pure silk fibroin scaffolds of A. mylitta. Chondrogenic differentiation of MSCs in the silk fibroin/chitosan and pure silk fibroin scaffolds was evident by real-time PCR analysis for cartilage-specific ECM gene markers. The results represent silk fibroin/chitosan blended 3D scaffolds as suitable scaffold for mesenchymal stem cells-based cartilage repair.     

Heparin crosslinked chitosan microspheres for the delivery of neural stem cells and growth factors for central nervous system repair

An effective paradigm for transplanting large numbers of neural stem cells after central nervous system (CNS) injury has yet to be established. Biomaterial scaffolds have shown promise in cell transplantation and in regenerative medicine, but improved scaffolds are needed. In this study we designed and optimized multifunctional and biocompatible chitosan-based films and microspheres for the delivery of neural stem cells and growth factors for CNS injuries. The chitosan microspheres were fabricated by coaxial airflow techniques, with the sphere size controlled by varying the syringe needle gauge and the airflow rate. When applying a coaxial airflow at 30 standard cubic feet per hour, ～300 μm diameter spheres were reproducibly generated that were physically stable yet susceptible to enzymatic degradation. Heparin was covalently crosslinked to the chitosan scaffolds using genipin, which bound fibroblast growth factor-2 (FGF-2) with high affinity while retaining its biological activity. At 1 μg ml^?1 approximately 80% of the FGF-2 bound to the scaffold. A neural stem cell line, GFP + RG3.6 derived from embryonic rat cortex, was used to evaluate cytocompatibility, attachment and survival on the crosslinked chitosan–heparin complex surfaces. The MTT assay and microscopic analysis revealed that the scaffold containing tethered FGF-2 was superior in sustaining survival and growth of neural stem cells compared to standard culture conditions. Altogether, our results demonstrate that this multifunctional scaffold possesses good cytocompatibility and can be used as a growth factor delivery vehicle while supporting neural stem cell attachment and survival.     

Adhesion,migration and mechanics of human adipose-tissue-derived stem cells on silk fibroin–chitosan matrix

Silk fibroin–chitosan (SFCS) scaffold is a naturally derived biocompatible matrix with potential reconstructive surgical applications. In this study, human adipose-derived mesenchymal stem cells (ASCs) were seeded on SFCS scaffolds and cell attachment was characterized by fluorescence, confocal, time-lapse, atomic force, and scanning electron microscopy (SEM) studies. Adhesion of ASCs on SFCS was 39.4 ± 4.8% at 15 min, increasing to 92.8 ± 1.5% at 120 min. ASC adhered at regions of architectural complexity and infiltrate into three-dimensional scaffold. Time-lapse confocal studies indicated a mean ASC speed on SFCS of 18.47 ± 2.7 μm h^?1 and a mean persistence time of 41.4 ± 9.3 min over a 2.75 h study period. Cytokinetic and SEM studies demonstrated ASC–ASC interaction via microvillus extensions. The apparent elastic modulus was significantly higher (p < 0.0001) for ASCs seeded on SFCS (69.0 ± 9.0 kPa) than on glass (6.1 ± 0.4 kPa). Also, cytoskeleton F-actin fiber density was higher (p < 0.05) for ASC seeded on SFCS (0.42 ± 0.02 fibers μm^?1) than on glass-seeded controls (0.24 ± 0.03 fibers μm^?1). Hence, SFCS scaffold facilitates mesenchymal stem cell attachment, migration, three-dimensional infiltration, and cell–cell interaction. This study showed the potential use of SFCS as a local carrier for autologous stem cells for reconstructive surgery application.     

Electrospinning and crosslinking of low-molecular-weight poly(trimethylene carbonate-co-l-lactide) as an elastomeric scaffold for vascular engineering

The growth of suitable tissue to replace natural blood vessels requires a degradable scaffold material that is processable into porous structures with appropriate mechanical and cell growth properties. This study investigates the fabrication of degradable, crosslinkable prepolymers of l-lactide-co-trimethylene carbonate into porous scaffolds by electrospinning. After crosslinking by γ-radiation, dimensionally stable scaffolds were obtained with up to 56% trimethylene carbonate incorporation. The fibrous mats showed Young’s moduli closely matching human arteries (0.4–0.8 MPa). Repeated cyclic extension yielded negligible change in mechanical properties, demonstrating the potential for use under dynamic physiological conditions. The scaffolds remained elastic and resilient at 30% strain after 84 days of degradation in phosphate buffer, while the modulus and ultimate stress and strain progressively decreased. The electrospun mats are mechanically superior to solid films of the same materials. In vitro, human mesenchymal stem cells adhered to and readily proliferated on the three-dimensional fiber network, demonstrating that these polymers may find use in growing artificial blood vessels in vivo.     

Biomimetic chitosan–nanohydroxyapatite composite scaffolds for bone tissue engineering

We describe a comparative assessment of the structure–property–process relationship of three-dimensional chitosan–nanohydroxyapatite (nHA) and pure chitosan scaffolds in conjunction with their respective biological response with the aim of advancing our insight into aspects that concern bone tissue engineering. High- and medium-molecular-weight (MW) chitosan scaffolds with 0.5, 1 and 2 wt.% fraction of nHA were fabricated by freezing and lyophilization. The nanocomposites were characterized by a highly porous structure and the pore size (～50 to 120 μm) was in a similar range for the scaffolds with different content of nHA. A combination of X-ray diffraction, Fourier transform infrared spectroscopy and electron microscopy indicated that nHA particles were uniformly dispersed in chitosan matrix and there was a chemical interaction between chitosan and nHA. The compression modulus of hydrated chitosan scaffolds was increased on the addition of 1 wt.% nHA from 6.0 to 9.2 kPa in high-MW scaffold. The water uptake ability of composites decreased with an increase in the amount of nHA, while the water retention ability was similar to pure chitosan scaffold. After 28 days in physiological condition, nanocomposites indicated about 10% lower degree of degradation in comparison to chitosan scaffold. The biological response of pre-osteoblasts (MC 3T3-E1) on nanocomposite scaffolds was superior in terms of improved cell attachment, higher proliferation, and well-spread morphology in relation to chitosan scaffold. In composite scaffolds, cell proliferation was about 1.5 times greater than pure chitosan after 7 days of culture and beyond, as implied by qualitative analysis via fluorescence microscopy and quantitative study through MTT assay. The observations related to well-developed structure morphology, physicochemical properties and superior cytocompatibility suggest that chitosan–nHA porous scaffolds are potential candidate materials for bone regeneration although it is necessary to further enhance the mechanical properties of the nanocomposite.     

Incorporation of bioactive polyvinylpyrrolidone–iodine within bilayered collagen scaffolds enhances the differentiation and subchondral osteogenesis of mesenchymal stem cells

Polyvinylpyrrolidone–iodine (Povidone-iodine, PVP-I) is widely used as an antiseptic agent for lavation during joint surgery; however, the biological effects of PVP–I on cells from joint tissue are unknown. This study examined the biocompatibility and biological effects of PVP–I on cells from joint tissue, with the aim of optimizing cell-scaffold based joint repair. Cells from joint tissue, including cartilage derived progenitor cells (CPC), subchondral bone derived osteoblast and bone marrow derived mesenchymal stem cells (BM-MSC) were isolated. The concentration-dependent effects of PVP–I on cell proliferation, migration and differentiation were evaluated. Additionally, the efficacy and mechanism of a PVP–I loaded bilayer collagen scaffold for osteochondral defect repair was investigated in a rabbit model. A micromolar concentration of PVP–I was found not to affect cell proliferation, CPC migration or extracellular matrix production. Interestingly, micromolar concentrations of PVP–I promote osteogenic differentiation of BM-MSC, as evidenced by up-regulation of RUNX2 and Osteocalcin gene expression, as well as increased mineralization on the three-dimensional scaffold. PVP–I treatment of collagen scaffolds significantly increased fibronectin binding onto the scaffold surface and collagen type I protein synthesis of cultured BM-MSC. Implantation of PVP–I treated collagen scaffolds into rabbit osteochondral defect significantly enhanced subchondral bone regeneration at 6 weeks post-surgery compared with the scaffold alone (subchondral bone histological score of 8.80 ± 1.64 vs. 3.8 ± 2.19, p < 0.05). The biocompatibility and pro-osteogenic activity of PVP–I on the cells from joint tissue and the enhanced subchondral bone formation in PVP–I treated scaffolds would thus indicate the potential of PVP–I for osteochondral defect repair.     

Human bone marrow stem cell-encapsulating calcium phosphate scaffolds for bone repair

Due to its injectability and excellent osteoconductivity, calcium phosphate cement (CPC) is highly promising for orthopedic applications. However, a literature search revealed no report on human bone marrow mesenchymal stem cell (hBMSC) encapsulation in CPC for bone tissue engineering. The aim of this study was to encapsulate hBMSCs in alginate hydrogel beads and then incorporate them into CPC, CPC–chitosan and CPC–chitosan–fiber scaffolds. Chitosan and degradable fibers were used to mechanically reinforce the scaffolds. After 21 days, that the percentage of live cells and the cell density of hBMSCs inside CPC-based constructs matched those in alginate without CPC, indicating that the CPC setting reaction did not harm the hBMSCs. Alkaline phosphate activity increased by 8-fold after 14 days. Mineral staining, scanning electron microscopy and X-ray diffraction confirmed that apatitic mineral was deposited by the cells. The amount of hBMSC-synthesized mineral in CPC–chitosan–fiber matched that in CPC without chitosan and fibers. Hence, adding chitosan and fibers, which reinforced the CPC, did not compromise hBMSC osteodifferentiation and mineral synthesis. In conclusion, hBMSCs were encapsulated in CPC and CPC–chitosan–fiber scaffolds for the first time. The encapsulated cells remained viable, osteodifferentiated and synthesized bone minerals. These self-setting, hBMSC-encapsulating CPC-based constructs may be promising for bone tissue engineering applications.     

An anti-inflammatory cell-free collagen/resveratrol scaffold for repairing osteochondral defects in rabbits

Inflammatory factor overexpression is the major cause of cartilage and osteochondral damage. Resveratrol (Res) is known for its anti-inflammatory, antioxidant and immunmodulatory properties. However, these effects are hampered by its water insolubility and rapid metabolism in vivo. To optimize its therapeutic efficacy in this study, Res was grafted to polyacrylic acid (PAA, 1000 Da) to obtain a macromolecular drug, PAA-Res, which was then incorporated into atelocollagen (Coll) hydrogels to fabricate anti-inflammatory cell-free (Coll/Res) scaffolds with improved mechanical strengths. The Coll/Res scaffolds demonstrated the ability to capture diphenylpicrylhydrazyl free radicals. Both pure Coll and Coll/Res scaffolds could maintain their original shape for 6 weeks in phosphate buffered saline. The scaffolds were degraded by collagenase over several days, and the degradation rate was slowed down by Res loading. The Coll and Coll/Res scaffolds with excellent cytocompatibility were shown to promote the proliferation and maintain the normal phenotype of the seeded chondrocytes and bone marrow stromal stem cells (BMSCs). In addition, the Coll/Res scaffold exhibited the capacity to protect the chondrocytes and BMSCs against reactive oxygen species. The acellular Coll/Res scaffolds were transplanted into the rabbit osteochondral defects. After implantation for 2, 4 and 6 weeks, the samples were retrieved for quantitative real-time polymerase chain reaction, and the inflammatory related genes interleukin-1β, matrix metalloproteinases-13, COX-2 and bone and cartilage related genes SOX-9, aggrecan, Coll II and Coll I were determined. Compared with the untreated defects, the inflammatory related genes were down-regulated and those bone and cartilage related genes were up-regulated by filling the defect with an anti-inflammatory scaffold. After 12 weeks, the osteochondral defects were completely repaired by the Coll/Res scaffold, and the neo-cartilage integrated well with its surrounding tissue and subchondral bone. Immunohistochemical and glycosaminoglycan staining confirmed the distribution of Coll II and glycosaminoglycans in the regenerated cartilage. The anti-inflammatory acellular Coll/Res scaffolds are convenient to administer in vivo, holding a greater potential for future clinical applications.     

A multilayered scaffold of a chitosan and gelatin hydrogel supported by a PCL core for cardiac tissue engineering

A three-dimensional scaffold composed of self-assembled polycaprolactone (PCL) sandwiched in a gelatin–chitosan hydrogel was developed for use as a biodegradable patch with a potential for surgical reconstruction of congenital heart defects. The PCL core provides surgical handling, suturability and high initial tensile strength, while the gelatin–chitosan scaffold allows for cell attachment, with pore size and mechanical properties conducive to cardiomyocyte migration and function. The ultimate tensile stress of the PCL core, made from blends of 10, 46 and 80 kDa (Mn) PCL, was controllable in the range of 2–4 MPa, with lower average molecular weight PCL blends correlating with lower tensile stress. Blends with lower molecular weight PCL also had faster degradation (controllable from 0% to 7% weight loss in saline over 30 days) and larger pores. PCL scaffolds supporting a gelatin–chitosan emulsion gel showed no significant alteration in tensile stress, strain or tensile modulus. However, the compressive modulus of the composite tissue was similar to that of native tissue (～15 kPa for 50% gelatin and 50% chitosan). Electron microscopy revealed that the gelatin–chitosan gel had a three-dimensional porous structure, with a mean pore diameter of ～80 μm, showed migration of neonatal rat ventricular myocytes (NRVM), maintained NRVM viability for over 7 days, and resulted in spontaneously beating scaffolds. This multi-layered scaffold has sufficient tensile strength and surgical handling for use as a cardiac patch, while allowing migration or pre-loading of cardiac cells in a biomimetic environment to allow for eventual degradation of the patch and incorporation into native tissue.     

Modified PHBV scaffolds by in situ UV polymerization: Structural characteristic,mechanical properties and bone mesenchymal stem cell compatibility

An ideal scaffold provides an interface for cell adhesion and maintains enough biomechanical support during tissue regeneration. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) scaffolds with pore sizes ranging from 100 to 500 μm and porosity ～90% were prepared by the particulate-leaching method, and then modified by the introduction of polyacrylamide (PAM) on the inner surface of scaffolds using in situ UV polymerization, with the aim of enhancing the biological and mechanical properties of the PHBV scaffolds. The modified PHBV scaffolds had interconnected pores with porosity of 75.4–78.6% and pore sizes at peak volume from 20 to 50 μm. The compressive load and modulus were up to 62.45 N and 1.06 MPa, respectively. The water swelling percentage (WSP) of the modified PHBV scaffolds increased notably compared with that of the PHBV scaffolds, with the maximum WSP at 537%. Sheep bone mesenchymal stem cells (BMSC) were cultured on the PHBV and modified PHBV. The hydrophilic PAM chains did not influence BMSC viability or proliferation index, but the initial cell adhesion at 1 h of culture was enhanced significantly. Framing PHBV scaffold along with gel-like PAM chains inside is a novel model of inner surface modification for PHBV scaffolds, which shows potential in tissue engineering applications.     

Effects of hydroxyapatite on endothelial network formation in collagen/fibrin composite hydrogels in vitro and in vivo

Co-culture of endothelial cells (EC) and mesenchymal stem cells (MSC) results in robust vascular network formation in constrained 3-D collagen/fibrin (COL/FIB) composite hydrogels. However, the ability to form endothelial networks is lost when such gels are allowed to compact via cell-mediated remodeling. In this study, we created co-cultures of human EC and human MSC in both constrained and unconstrained COL/FIB matrices and systematically added nanoparticulate hydroxyapatite (HA, 0–20 mg ml⁻¹), a bone-like mineral that has been shown to have pro-vasculogenic effects. Constructs cultured for 7 days were assayed for gel compaction, vascular network formation, and mechanical properties. In vitro, robust endothelial network formation was observed in constrained COL/FIB constructs without HA, but this response was significantly inhibited by addition of 5, 10, or 20 mg ml⁻¹ HA. In unconstrained matrices, network formation was abolished in pure COL/FIB constructs but was rescued by 1.25 or 2.5 mg ml⁻¹ HA, while higher levels again inhibited vasculogenesis. HA inhibited gel compaction in a dose-dependent manner, which was not correlated to endothelial network formation. HA affected initial stiffness of the gels, but gel remodeling abrogated this effect. Subcutaneous implantation of COL/FIB with 0, 2.5 or 20 mg ml⁻¹ HA in the mouse resulted in increased perfusion at the implant site, with no significant differences between materials. Histology at day 7 showed both host and human CD31-stained vasculature infiltrating the implants. These findings are relevant to the design of materials and scaffolds for orthopedic tissue engineering, where both vasculogenesis and formation of a mineral phase are required for regeneration.     

Mesenchymal stem cell proliferation and differentiation on an injectable calcium phosphate – Chitosan composite scaffold

Calcium phosphate cement (CPC) can be molded or injected to form a scaffold in situ, has excellent osteoconductivity, and can be resorbed and replaced by new bone. However, its low strength limits CPC to non-stress-bearing repairs. Chitosan could be used to reinforce CPC, but mesenchymal stem cell (MSC) interactions with CPC-chitosan scaffold have not been examined. The objective of this study was to investigate MSC proliferation and osteogenic differentiation on high-strength CPC-chitosan scaffold. MSCs were harvested from rat bone marrow. At CPC powder/liquid (P/L) mass ratio of 2, flexural strength (mean ± sd; n = 5) was (10.0 ± 1.1) MPa for CPC-chitosan, higher than (3.7 ± 0.6) MPa for CPC (p < 0.05). At P/L of 3, strength was (15.7 ± 1.7) MPa for CPC-chitosan, higher than (10.2 ± 1.8) MPa for CPC (p < 0.05). Percentage of live MSCs attaching to scaffolds increased from 85% at 1 day to 99% at 14 days. There were (180 ± 37) cells/mm² on scaffold at 1 day; cells proliferated to (1808 ± 317) cells/mm² at 14 days. SEM showed MSCs with healthy spreading and anchored on nano-apatite crystals via cytoplasmic processes. Alkaline phosphatase activity (ALP) was (557 ± 171) (pNPP mM/min)/(μg DNA) for MSCs on CPC-chitosan, higher than (159 ± 47) on CPC (p < 0.05). Both were higher than (35 ± 32) of baseline ALP for undifferentiated MSCs on tissue-culture plastic (p < 0.05). In summary, CPC-chitosan scaffold had higher strength than CPC. MSC proliferation on CPC-chitosan matched that of the FDA-approved CPC control. MSCs on the scaffolds differentiated down the osteogenic lineage and expressed high levels of bone marker ALP. Hence, the stronger CPC-chitosan scaffold may be useful for stem cell-based bone regeneration in moderate load-bearing maxillofacial and orthopedic applications.     

Non-viral endostatin plasmid transfection of mesenchymal stem cells via collagen scaffolds

Angiogenesis is critical in the early stage of reparative processes and tissue regeneration, but the persistence of a vascular network may interfere with later transformation/maturation in naturally avascular tissues such as articular cartilage. Our supposition is that the timed delivery of an anti-angiogenic factor in cartilage tissue engineering may facilitate the formation of hyaline cartilage by inducing the regression of vascularization. To this end our overall goal is to prepare an off-the-shelf scaffold containing the gene for a potent anti-angiogenic factor. The objective of this study was to investigate the use of a type I/III collagen scaffold for the non-viral transfection of marrow stromal cells (MSCs, also referred to as mesenchymal stem cells) with the plasmid encoding endostatin. Caprine MSCs were transfected by the naked plasmid alone and plasmid incorporated into a cationic lipid complex in three experiments: 1) cells were transfected in monolayer; 2) monolayer-transfected cells were grown in a collagen sponge-like scaffold; and 3) non-transfected cells were grown in a collagen scaffold containing the naked plasmid and endostatin lipoplex. Independent variables were the passage number of the cells and the plasmid loading. The amount of endostatin released by the cells into the medium was measured using an ELISA. The results demonstrated the overexpression of endostatin by MSCs growing in the endostatin lipoplex-supplemented collagen scaffolds. Endostatin released by the cell-seeded scaffolds reached a peak of 13 ng/ml for scaffolds incorporating as little as 20 μg of plasmid, at the 3-day collection period ending 5 days post-seeding. The accumulated endostatin synthesis over a 2-week period began to achieve what may be a therapeutic level. MSCs transfected with the endostatin gene in monolayer continued to express the gene when grown in the collagen scaffolds. The results demonstrate the promise of the non-viral delivery of the gene for this potent anti-angiogenic protein to MSCs via a collagen scaffold.     

Biodegradable fibrous scaffolds with diverse properties by electrospinning candidates from a combinatorial macromer library

The properties of electrospun fibrous scaffolds, including degradation, mechanics and cellular interactions, are important for their use in tissue engineering applications. Although some diversity has been obtained previously in fibrous scaffolds, optimization of scaffold properties relies on iterative techniques in both polymer synthesis and processing. Here, we electrospun candidates from a combinatorial library of biodegradable and photopolymerizable poly(β-amino ester)s (PBAEs) to show that the diversity in properties found in this library is retained when processed into fibrous scaffolds. Specifically, three PBAE macromers were electrospun into scaffolds and possessed similar initial mechanical properties, but exhibited mass loss ranging from rapid (complete degradation within ～2 weeks) to moderate (complete degradation within ～3 months) to slow (only partial degradation after 3 months). These trends in mechanics and degradation mimicked what was previously observed in the bulk polymers. Although cellular adhesion was dependent on the polymer composition in films, adhesion to scaffolds that were electrospun with gelatin was similar on all formulations and controls. To further illustrate the diverse properties that are attainable in these systems, the fastest and slowest degrading polymers were electrospun together into one scaffold, but as distinct fiber populations. This dual-polymer scaffold exhibited behavior in mass loss and mechanics with time that fell between the single-polymer scaffolds. In general, this work indicates that combinatorial libraries may be an important source of information and specific polymer compositions for the fabrication of electrospun fibrous scaffolds with tunable properties.     

Mesenchymal stem cell-collagen microspheres for articular cartilage repair: Cell density and differentiation status

Mesenchymal stem cells (MSC) hold promise for cartilage repair. A microencapsulation technique was previously established to entrap MSC in collagen microspheres, and the collagen fibrous meshwork was found to be an excellent scaffold for supporting MSC survival, growth and differentiation. This study investigates the importance of cell density and differentiation status of MSC–collagen microspheres in cartilage repair. MSC were isolated from rabbit bone marrow and encapsulated in collagen microspheres. The effects of pre-differentiating the encapsulated MSC into chondrogenic lineages and different cell densities on cartilage repair were investigated in rabbits. Implantation of undifferentiated collagen–MSC microspheres formed hyaline-like cartilage rich in type II collagen and glycosaminoglycans (GAG) at 1 month post-implantation. By 6 months, hyaline cartilage rich in type II collagen and GAG, but negative for type I collagen, and partial zonal organization were found in both undifferentiated and chondrogenically differentiated groups in the high cell density group. The undifferentiated group and high cell density group significantly improved the O’Driscoll histological score. Moreover, the undifferentiated group significantly increased the GAG content. The mechanically differentiated group showed stiffer but thinner cartilage, while the undifferentiated group showed thicker but softer cartilage compared with their respective contra-lateral controls. This work suggests that a higher local cell density favors cartilage regeneration, regardless of the differentiation status of MSC, while the differentiation status of MSC does significantly affect regeneration outcomes.