Cartilage graft engineering by co‐culturing primary human articular chondrocytes with human bone marrow stromal cells

Co‐culture of mesenchymal stromal cells (MSCs) with articular chondrocytes (ACs) has been reported to improve the efficiency of utilization of a small number of ACs for the engineering of implantable cartilaginous tissues. However, the use of cells of animal origin and the generation of small‐scale micromass tissues limit the clinical relevance of previous studies. Here we investigated the in vitro and in vivo chondrogenic capacities of scaffold‐based constructs generated by combining primary human ACs with human bone marrow MSCs (BM‐MSCs). The two cell types were cultured in collagen sponges (2 × 6 mm disks) at the BM‐MSCs:ACs ratios: 100:0, 95:5, 75:25 and 0:100 for 3 weeks. Scaffolds freshly seeded or further precultured in vitro for 2 weeks were also implanted subcutaneously in nude mice and harvested after 8 or 6 weeks, respectively. Static co‐culture of ACs (25%) with BM‐MSCs (75%) in scaffolds resulted in up to 1.4‐fold higher glycosaminoglycan (GAG) content than what would be expected based on the relative percentages of the different cell types. In vivo GAG induction was drastically enhanced by the in vitro preculture and maximal at the ratio 95:5 (3.8‐fold higher). Immunostaining analyses revealed enhanced accumulation of type II collagen and reduced accumulation of type X collagen with increasing ACs percentage. Constructs generated in the perfusion bioreactor system were homogeneously cellularized. In summary, human cartilage grafts were successfully generated, culturing BM‐MSCs with a relatively low fraction of non‐expanded ACs in porous scaffolds. The proposed co‐culture strategy is directly relevant towards a single‐stage surgical procedure for cartilage repair. Copyright © 2012 John Wiley & Sons, Ltd.     

The differential in vitro and in vivo responses of bone marrow stromal cells on novel porous gelatin–alginate scaffolds

Tissue engineering and stem cell therapy hold great potential of being able to fully restore, repair and replace damaged, diseased or lost tissues in the body. Biocompatible porous scaffolds are used for the delivery of cells to the regeneration sites. Marrow stromal cells (MSCs), also referred to as mesenchymal stem cells, are an attractive cell source for tissue engineering, due to the relative ease of isolation and the ability of in vitro expanded MSCs to generate multiple cell types, including osteoblasts, chondrocytes and adipocytes. This study utilized a novel technique called microwave vacuum drying to fabricate porous gelatin–alginate scaffolds for the delivery of MSCs and investigated the differential in vitro and in vivo responses of MSCs seeded on these scaffolds. Scaffold total porosity was found to decrease with increased cross‐link density but the pore size and pore size distribution were not affected. Although highly porous, the scaffold had relatively small pores and limited interconnectivity. The porous gelatin–alginate scaffold demonstrated excellent biocompatibility with neovascularization on the surfaces and was bioresorbed completely in vivo, depending upon the cross‐link density. MSCs were able to attach and proliferate at the same rate on the scaffolds, and the self‐renewal potential of MSC cultures was similar during both in vitro culture and in vivo implantation. However, the subcutaneous microenvironment was found to suppress MSC differentiation along the osteogenic, chondrogenic and adipogenic lineages compared to in vitro conditions, highlighting the differential responses of MSCs cultured in vitro compared to implantation in vivo. Copyright © 2009 John Wiley & Sons, Ltd.     

Modulation of chondrogenic differentiation of human mesenchymal stem cells in jellyfish collagen scaffolds by cell density and culture medium

Studies on tissue‐engineering approaches for the regeneration of traumatized cartilage focus increasingly on multipotent human mesenchymal stem cells (hMSCs) as an alternative to autologous chondrocytes. The present study applied porous scaffolds made of collagen from the jellyfish Rhopilema esculentum for the in vitro chondrogenic differentiation of hMSCs. Culture conditions in those scaffolds differ from conditions in high‐density pellet cultures, making a re‐examination of these data necessary. We systematically investigated the influence of seeding density, basic culture media [Dulbecco's modified Eagle's medium (DMEM), α‐minimum essential medium (α‐MEM)] with varying glucose content and supplementation with fetal calf serum (FCS) or bovine serum albumin (BSA) on the chondrogenic differentiation of hMSCs. Gene expression analyses of selected markers for chondrogenic differentiation and hypertrophic development were conducted. Furthermore, the production of cartilage extracellular matrix (ECM) was analysed by quantification of sulphated glycosaminoglycan and collagen type II contents. The strongest upregulation of chondrogenic markers, along with the highest ECM deposition was observed in scaffolds seeded with 2.4 × 10⁶ cells/cm³ after cultivation in high‐glucose DMEM and 0.125% BSA. Lower seeding densities compared to high‐density pellet cultures were sufficient to induce in vitro chondrogenic differentiation of hMSCs in collagen scaffolds, which reduces the amount of cells required for the seeding of scaffolds and thus the monolayer expansion period. Furthermore, examination of the impact of FCS and α‐MEM on chondrogenic MSC differentiation is an important prerequisite for the development of an osteochondral medium for simultaneous osteogenic and chondrogenic differentiation in biphasic scaffolds for osteochondral tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Investigation of the optimal timing for chondrogenic priming of MSCs to enhance osteogenic differentiation in vitro as a bone tissue engineering strategy

Recent in vitro tissue engineering approaches have shown that chondrogenic priming of human bone marrow mesenchymal stem cells (MSCs) can have a positive effect on osteogenesis in vivo. However, whether chondrogenic priming is an effective in vitro bone regeneration strategy is not yet known. In particular, the appropriate timing for chondrogenic priming in vitro is unknown albeit that in vivo cartilage formation persists for a specific period before bone formation. The objective of this study is to determine the optimum time for chondrogenic priming of MSCs to enhance osteogenic differentiation by MSCs in vitro. Pellets derived from murine and human MSCs were cultured in six different media groups: two control groups (chondrogenic and osteogenic) and four chondrogenic priming groups (10, 14, 21 and 28 days priming). Biochemical analyses (Hoechst, sulfate glycosaminoglycan (sGAG), Alkaline Phosphate (ALP), calcium), histology (Alcian Blue, Alizarin Red) and immunohistochemistry (collagen types I, II and X) were performed on the samples at specific times. Our results show that after 49 days the highest amount of sGAG production occurred in MSCs chondrogenically primed for 21 days and 28 days. Moreover we found that chondrogenic priming of MSCs in vitro for specific amounts of time (14 days, 21 days) can have optimum influence on their mineralization capacity and can produce a construct that is mineralized throughout the core. Determining the optimum time for chondrogenic priming to enhance osteogenic differentiation in vitro provides information that might lead to a novel regenerative treatment for large bone defects, as well as addressing the major limitation of core degradation and construct failure. Copyright © 2013 John Wiley & Sons, Ltd.     

Efficacy of thermoresponsive,photocrosslinkable hydrogels derived from decellularized tendon and cartilage extracellular matrix for cartilage tissue engineering

Tissue engineering using adult mesenchymal stem cells (MSCs), a promising approach for cartilage repair, is highly dependent on the nature of the matrix scaffold. Thermoresponsive, photocrosslinkable hydrogels were fabricated by functionalizing pepsin‐soluble decellularized tendon and cartilage extracellular matrices (ECM) with methacrylate groups. Methacrylated gelatin hydrogels served as controls. When seeded with human bone marrow MSCs and cultured in chondrogenic medium, methacrylated ECM hydrogels experienced less cell‐mediated contraction, as compared against non‐methacrylated ECM hydrogels. However, methacrylation slowed or diminished chondrogenic differentiation of seeded MSCs, as determined through analyses of gene expression, biochemical composition and histology. In particular, methacrylated cartilage hydrogels supported minimal due to chondrogenesis over 42 weeks, as hydrogel disintegration beginning at day 14 presumably compromised cell–matrix interactions. As compared against methacrylated gelatin hydrogels, MSCs cultured in non‐methacrylated ECM hydrogels exhibited comparable expression of chondrogenic genes (Sox9, Aggrecan and collagen type II) but increased collagen type I expression. Non‐methacrylated cartilage hydrogels did not promote chondrogenesis to a greater extent than either non‐methacrylated or methacrylated tendon hydrogels. Whereas methacrylated gelatin hydrogels supported relatively homogeneous increases in proteoglycan and collagen type II deposition throughout the construct over 42 days, ECM hydrogels possessed greater heterogeneity of staining intensity and construct morphology. These results do not support the utility of pepsin‐solubilized cartilage and tendon hydrogels for cartilage tissue engineering over methacrylated gelatin hydrogels. Methacrylation of tendon and cartilage ECM hydrogels permits thermal‐ and light‐induced polymerization but compromises chondrogenic differentiation of seeded MSCs.     

Ectopic models for endochondral ossification: comparing pellet and alginate bead culture methods

Key aspects of native endochondral bone development and fracture healing can be mimicked in mesenchymal stem cells (MSCs) through standard in vitro chondrogenic induction. Exploiting this phenomenon has recently emerged as an attractive technique to engineer bone tissue, however, relatively little is known about the best conditions for doing so. The objective of the present study was to compare the bone‐forming capacity and angiogenic induction of hypertrophic cell constructs containing human adipose‐derived stem cells (hASCs) primed for chondrogenesis in two different culture systems: high‐density pellets and alginate bead hydrogels. The hASC constructs were subjected to 4 weeks of identical chondrogenic induction in vitro, encapsulated in an agarose carrier, and then implanted subcutaneously in immune‐compromised mice for 8 weeks to evaluate their endochondral potential. At the time of implantation, both pellets and beads expressed aggrecan and type II collagen, as well as alkaline phosphatase (ALP) and type X collagen. Interestingly, ASCs in pellets formed a matrix containing higher glycosaminoglycan and collagen contents than that in beads, and ALP activity per cell was higher in pellets. However, after 8 weeks in vivo, pellets and beads induced an equivalent volume of mineralized tissue and a comparable level of vascularization. Although osteocalcin and osteopontin‐positive osteogenic tissue and new vascular growth was found within both types of constructs, all appeared to be better distributed throughout the hydrogel beads. The results of this ectopic model indicate that hydrogel culture may be an attractive alternative to cell pellets for bone tissue engineering via the endochondral pathway. Copyright © 2016 John Wiley & Sons, Ltd.     

Novel injectable gel (system) as a vehicle for human articular chondrocytes in cartilage tissue regeneration

We developed a novel injectable carrageenan/fibrin/hyaluronic acid‐based hydrogel with in situ gelling properties to be seeded with chondrogenic cells and used for cartilage tissue engineering applications. We first analysed the distribution within the hydrogel construct and the phenotype of human articular chondrocytes (HACs) cultured for 3 weeks in vitro. We observed a statistically significant increase in the cell number during the first 2 weeks and maintenance of cell viability throughout the cell culture, together with the deposition/formation of a cartilage‐specific extracellular matrix (ECM). Taking advantage of a new in vivo model that allows the integration between newly formed and preexisting cartilage in immunodeficient mice to be investigated, we showed that injectable hydrogel seeded with human articular chondrocytes was able to regenerate and repair an experimentally made lesion in bovine articular cartilage, thus demonstrating the potential of this novel cell delivery system for cartilage tissue engineering. Copyright © 2009 John Wiley & Sons, Ltd.     

Evaluation of articular cartilage repair using biodegradable nanofibrous scaffolds in a swine model: a pilot study

The aim of this study was to evaluate a cell‐seeded nanofibrous scaffold for cartilage repair in vivo. We used a biodegradable poly(ε‐caprolactone) (PCL) nanofibrous scaffold seeded with allogeneic chondrocytes or xenogeneic human mesenchymal stem cells (MSCs), or acellular PCL scaffolds, with no implant as a control to repair iatrogenic, 7 mm full‐thickness cartilage defects in a swine model. Six months after implantation, MSC‐seeded constructs showed the most complete repair in the defects compared to other groups. Macroscopically, the MSC‐seeded constructs regenerated hyaline cartilage‐like tissue and restored a smooth cartilage surface, while the chondrocyte‐seeded constructs produced mostly fibrocartilage‐like tissue with a discontinuous superficial cartilage contour. Incomplete repair containing fibrocartilage or fibrous tissue was found in the acellular constructs and the no‐implant control group. Quantitative histological evaluation showed overall higher scores for the chondrocyte‐ and MSC‐seeded constructs than the acellular construct and the no‐implant groups. Mechanical testing showed the highest equilibrium compressive stress of 1.5 MPa in the regenerated cartilage produced by the MSC‐seeded constructs, compared to 1.2 MPa in the chondrocyte‐seeded constructs, 1.0 MPa in the acellular constructs and 0.2 MPa in the no‐implant group. No evidence of immune reaction to the allogeneically‐ and xenogeneically‐derived regenerated cartilage was observed, possibly related to the immunosuppressive activities of MSCs, suggesting the feasibility of allogeneic or xenogeneic transplantation of MSCs for cell‐based therapy. Taken together, our results showed that biodegradable nanofibrous scaffolds seeded with MSCs effectively repair cartilage defects in vivo, and that the current approach is promising for cartilage repair. Copyright © 2008 John Wiley & Sons, Ltd.     

Chondrogenic Differentiation Increases Antidonor Immune Response to Allogeneic Mesenchymal Stem Cell Transplantation

Allogeneic mesenchymal stem cells (allo-MSCs) have potent regenerative and immunosuppressive potential and are being investigated as a therapy for osteoarthritis; however, little is known about the immunological changes that occur in allo-MSCs after ex vivo induced or in vivo differentiation. Three-dimensional chondrogenic differentiation was induced in an alginate matrix, which served to immobilize and potentially protect MSCs at the site of implantation. We show that allogeneic differentiated MSCs lost the ability to inhibit T-cell proliferation in vitro, in association with reduced nitric oxide and prostaglandin E2 secretion. Differentiation altered immunogenicity as evidenced by induced proliferation of allogeneic T cells and increased susceptibility to cytotoxic lysis by allo-specific T cells. Undifferentiated or differentiated allo-MSCs were implanted subcutaneously, with and without alginate encapsulation. Increased CD3⁺ and CD68⁺ infiltration was evident in differentiated and splenocyte encapsulated implants only. Without encapsulation, increased local memory T-cell responses were detectable in recipients of undifferentiated and differentiated MSCs; however, only differentiated MSCs induced systemic memory T-cell responses. In recipients of encapsulated allogeneic cells, only differentiated allo-MSCs induced memory T-cell responses locally and systemically. Systemic alloimmune responses to differentiated MSCs indicate immunogenicity regardless of alginate encapsulation and may require immunosuppressive therapy for therapeutic use.     

Asymmetrical seeding of MSCs into fibrin–poly(ester‐urethane) scaffolds and its effect on mechanically induced chondrogenesis

Mesenchymal stem cells (MSCs) are currently being investigated as candidate cells for regenerative medicine approaches for the repair of damaged articular cartilage. For these cells to be used clinically, it is important to understand how they will react to the complex loading environment of a joint in vivo. In addition to investigating alternative cell sources, it is also important for the structure of tissue‐engineered constructs and the organization of cells within them to be developed and, if possible, improved. A custom built bioreactor was used to expose human MSCs to a combination of shear and compression loading. The MSCs were either evenly distributed throughout fibrin‐poly(ester‐urethane) scaffolds or asymmetrically seeded with a small proportion seeded on the surface of the scaffold. The effect of cell distribution on the production and deposition of cartilage‐like matrix in response to mechanical load mimicking in vivo joint loading was then investigated. The results show that asymmetrically seeding the scaffold led to markedly improved tissue development based on histologically detectable matrix deposition. Consideration of cell location, therefore, is an important aspect in the development of regenerative medicine approaches for cartilage repair. This is particularly relevant when considering the natural biomechanical environment of the joint in vivo and patient rehabilitation protocols. © 2016 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons Ltd.     

Evaluation of platelet‐rich plasma and hydrostatic pressure regarding cell differentiation in nucleus pulposus tissue engineering

Generation of a biological nucleus pulposus (NP) replacement by tissue engineering appears to be a promising approach for the therapy of early stages of intervertebral disc degeneration. Thereby, autologous mesenchymal stem cells (MSCs) represent an attractive cell source compared to cells of the NP that are already altered in their phenotype due to degenerative processes. This study compares the influence of 3D pellet culture and alginate beads, as well as that of different media compositions, by the addition of human platelet‐rich plasma (PRP) or transforming growth factor (TGF‐β₁) in interaction with hydrostatic pressure on chondrogenic differentiation of human MSCs compared to NP cells. We found that gene expression of the chondrogenic markers aggrecan, collagen type 2 and collagen type 1 and Sox9 was considerably lower in cells cultivated with PRP compared to TGF‐β₁. Immunohistology confirmed this result at protein level in pellet culture. Additionally, the pellet culture system was found to be more suitable than alginate beads. A positive influence of hydrostatic pressure could only be shown for individual donors. In summary, in comparison to TGF‐β₁, human PRP did not induce adequate chondrogenic differentiation for both culture systems and cell types used. The mixture of growth factors in PRP promoted proliferation rather than chondrogenic differentiation. Based on these results, an application of PRP in human NP tissue‐engineering approaches cannot be recommended. Copyright © 2011 John Wiley & Sons, Ltd.     

Effect of chondroitin sulphate C on the in vitro and in vivo chondrogenesis of mesenchymal stem cells in crosslinked type II collagen scaffolds

This study evaluates the crosslinkage effect of chondroitin sulphate C (CSC) and type II collagen (COL II) on chondrogenesis of mesenchymal stem cells (MSCs) in vitro and in vivo. In the in vitro studies, our results show that the weight ratio CSC:COL II that reaches 1.2:100 (CSC_1.2/100–COL II scaffold) can provide an optimal microenvironment for MSC chondrogenesis. When MSCs are cultured in this CSC_1.2/100–COL II scaffold, the chondrogenic gene expression of cultured cells is upregulated, while the osteogenic gene expression of these is downregulated. In addition, MSCs cultivated in the CSC_1.2/100–COL II scaffold are found to express the highest glycosaminoglycans:DNA ratio as compared to those in scaffolds of other CSC:COL II ratios. Histological and immunohistological evidence also supports the result. In the in vivo study, our results show that MSCs cultivated in the CSC_1.2/100–COL II scaffold demonstrate a better repair ability on cartilage lesions than does the COL II scaffold. After 1 month in vivo, the injected MSCs in the CSC_1.2/100–COL II scaffold show lacuna structures and stimulate the formation of type II collagen at the defective sites. Six months after transplantation, the generated cells in the CSC_1.2/100–COL II group show higher gene expressions of type II collagen and aggrecan but lower gene expression of type I collagen at the defective sites than those in the COL II group. The results strongly suggest that CSC_1.2/100–COL II scaffold can serve as a potential candidate for cartilage repair and improve the chondrogenesis of MSCs in general. Copyright © 2012 John Wiley & Sons, Ltd.     

In vitro and in vivo co‐culture of chondrocytes and bone marrow stem cells in photocrosslinked PCL–PEG–PCL hydrogels enhances cartilage formation

Chondrocytes (CH) and bone marrow stem cells (BMSCs) are sources that can be used in cartilage tissue engineering. Co‐culture of CHs and BMSCs is a promising strategy for promoting chondrogenic differentiation. In this study, articular CHs and BMSCs were encapsulated in PCL–PEG–PCL photocrosslinked hydrogels for 4 weeks. Various ratios of CH:BMSC co‐cultures were investigated to identify the optimal ratio for cartilage formation. The results thus obtained revealed that co‐culturing CHs and BMSCs in hydrogels provides an appropriate in vitro microenvironment for chondrogenic differentiation and cartilage matrix production. Co‐culture with a 1:4 CH:BMSC ratio significantly increased the synthesis of GAGs and collagen. In vivo cartilage regeneration was evaluated using a co‐culture system in rabbit models. The co‐culture system exhibited a hyaline chondrocyte phenotype with excellent regeneration, resembling the morphology of native cartilage. This finding suggests that the co‐culture of these two cell types promotes cartilage regeneration and that the system, including the hydrogel scaffold, has potential in cartilage tissue engineering. Copyright © 2013 John Wiley & Sons, Ltd.     

Biomimetic scaffolds and dynamic compression enhance the properties of chondrocyte‐ and MSC‐based tissue‐engineered cartilage

Adult chondrocytes are surrounded by a protein‐ and glycosaminoglycan‐rich extracellular matrix and are subjected to dynamic mechanical compression during daily activities. The extracellular matrix and mechanical stimuli play an important role in chondrocyte biosynthesis and homeostasis. In this study, we aimed to develop scaffold and compressive loading conditions that mimic the native cartilage micro‐environment and enable enhanced chondrogenesis for tissue engineering applications. Towards this aim, we fabricated porous scaffolds based on silk fibroin (SF) and SF with gelatin/chondroitin sulfate/hyaluronate (SF‐GCH), seeded the scaffolds with either human bone marrow mesenchymal stromal cells (BM‐MSCs) or chondrocytes, and evaluated their performance with and without dynamic compression. Human chondrocytes derived from osteoarthritic joints and BM‐MSCs were seeded in scaffolds, precultured for 1 week, and subjected to compression with 10% dynamic strain at 1 Hz, 1 hr/day for 2 weeks. When dynamic compression was applied, chondrocytes significantly increased expression of aggrecan (ACAN) and collagen X (COL10A1) up to fivefold higher than free‐swelling controls. In addition, dynamic compression dramatically improved the chondrogenesis and chondrocyte biosynthesis cultured in both SF and SF‐GCH scaffolds evidenced by glycosaminoglycan (GAG) content, GAG/DNA ratio, and immunostaining of collagen type II and aggrecan. However, both chondrocytes and BM‐MSCs cultured in SF‐GCH scaffolds under dynamic compression showed higher GAG content and compressive modulus than those in SF scaffolds. In conclusion, the micro‐environment provided by SF‐GCH scaffolds and dynamic compression enhances chondrocyte biosynthesis and matrix accumulation, indicating their potential for cartilage tissue engineering applications.     

Treatment of growth plate injury using IGF‐I‐loaded PLGA scaffolds

Growth plate fracture can lead to retarded growth and unequal limb length, which may have a lifelong effect on a person's physical stature. The goal of this research was to develop an in vivo tissue‐engineering approach for the treatment of growth plate injury via localized delivery of insulin‐like growth factor I (IGF‐I) from cell‐free poly(lactic‐co‐glycolic acid) (PLGA) scaffolds. Mass loss and drug release studies were conducted to study the scaffold degradation and IGF‐I release patterns. In vitro cell studies showed that rat bone marrow stromal cells seeded on the porous scaffolds colonized the pores and deposited matrix within the scaffolds. These in vitro evaluations were followed by a proof‐of‐concept animal study involving implantation of scaffolds in proximal tibial growth plate defects in New Zealand white rabbits. Histological analysis of tissue sections from the in vivo studies showed regeneration of cartilage, albeit with disorganized structure, at the site of implantation of IGF‐I‐releasing scaffolds; in contrast, only bone was formed in empty defects and those treated with IGF‐free scaffolds. The present findings show the potential for treating growth plate injury using in vivo tissue engineering techniques. Copyright © 2012 John Wiley & Sons, Ltd.     

Ectopic bone formation by aggregated mesenchymal stem cells from bone marrow and adipose tissue: A comparative study

Tissue engineered constructs (TECs) based on spheroids of bone marrow mesenchymal stromal cells (BM‐MSCs) combined with calcium phosphate microparticles and enveloped in a platelet‐rich plasma hydrogel showed that aggregation of MSCs improves their ectopic bone formation potential. The stromal vascular fraction (SVF) and adipose‐derived MSCs (ASCs) have been recognized as an interesting MSC source for bone tissue engineering, but their ectopic bone formation is limited. We investigated whether aggregation of ASCs could similarly improve ectopic bone formation by ASCs and SVF cells. The formation of aggregates with BM‐MSCs, ASCs and SVF cells was carried out and gene expression was analysed for osteogenic, chondrogenic and vasculogenic genes in vitro. Ectopic bone formation was evaluated after implantation of TECs in immunodeficient mice with six conditions: TECs with ASCs, TECs with BM‐MSC, TECs with SVF cells (with and without rhBMP2), no cells and no cells with rhBMP2. BM‐MSCs showed consistent compact spheroid formation, ASCs to a lesser extent and SVF showed poor spheroid formation. Aggregation of ASCs induced a significant upregulation of the expression of osteogenic markers like alkaline phosphatase and collagen type I, as compared with un‐aggregated ASCs. In vivo, ASC and SVF cells both generated ectopic bone in the absence of added morphogenetic proteins. The highest incidence of bone formation was seen with BM‐MSCs (7/9) followed by SVF + rhBMP2 (4/9) and no cells + rhBMP2 (2/9). Aggregation can improve ectopic bone tissue formation by adipose‐derived cells, but is less efficient than rhBMP2. A combination of both factors should now be tested to investigate an additive effect.     

TGF-β3: A potential biological therapy for enhancing chondrogenesis

Background: TGF-β has been proposed to stimulate chondrogenesis through intracellular pathways involving small mothers against decapentaplegic proteins (Smads). Objective: To examine the use of exogenous TGF-β3 to promote new hyaline cartilage formation. Methods: An overview of in vitro and in vivo evidence on the effects of TGF-β3 on cartilage regeneration. Results/conclusion: There is robust in vitro evidence suggesting a positive dose- and time-dependant effect of TGF-β3 on anabolic chondrogenic gene markers such as α₁-collagen type II and cartilage oligomeric matrix protein in human mesenchymal stem cells. TGF-β3 cultured with silk elastin-like polymer scaffold carrier exhibits significantly increased glycosaminoglycan and collagen content. In vivo data showed that TGF-β3 cultured with ovine mesenchymal stem cells in a chitosan scaffold stimulated the growth of hyaline cartilage that was fully integrated into host cartilage tissue of sheep. We highlight the potential for the clinical enhancement of cartilage formation through the use of TGF-β3 with a suitable dose and scaffold carrier.     

Evaluation of cross‐linked chitosan microparticles for bone regeneration

The objective of this preliminary study was to evaluate the applying of chitosan (CS)‐based microparticles (MPs) in bone regeneration in vivo. The CS MPs were fabricated using our scale‐up method, as previously described. Mesenchymal stem cells (MSC) were harvested from the femora and tibiae of Dark Agouti (DA) rats and seeded on CS MPs. An in vitro MSCs attachment experiment was conducted by trypsinizing the cells attached to the MPs at 5, 10, 20 and 30 h. Fluorescence images of MSCs attached to the MPs were taken at 24 and 48 h, using a LIVE/DEAD cell assay. The MSC/osteoblasts (OB) seeded on MPs were then cultured in vitro using osteogenic media and implanted into partial thickness bone defects in rat femurs. There were two groups of rats, including experimental animals and controls, for the in vivo studies. The experimental group were implanted with MSC‐seeded MPs and observed at 4 and 8 weeks. The control group of rats did not receive any implant material except the stainless steel plate to support the defect. Four rats per group were used for the study. The femurs were extracted at 4 and 8 weeks post‐implantation and bone formation at the defect site was analysed using radiography, microcomputed tomography (µCT) and histology. Among all groups, a significant increase in bone formation was observed in the experimental group at 8 weeks implantation. The results of this study suggested that CS MPs prove to be a successful biomaterial for bone regeneration. Copyright © 2010 John Wiley & Sons, Ltd.