In vitro engineered cartilage using synovium-derived mesenchymal stem cells with injectable gellan hydrogels

Synovium-derived mesenchymal stem cells (SMSC), a novel line of stem cells, are regarded as a promising cell source for cartilage tissue engineering. The goal of this study was to investigate rabbit SMSC coupled with injectable gellan hydrogels for in vitro engineered cartilage. SMSC were isolated from rabbit synovial tissue, amplified to passage 4 in monolayer, and encapsulated in injectable gellan hydrogels, constructs of which were cultured in chondrogenic medium supplemented with TGF-β1, TGF-β3 or BMP-2 for up to 42 days. The quality of the constructs was assessed in terms of cell proliferation and chondrocytic gene/protein expression using WST-1 assay, real-time RT-PCR, biochemical analysis, histology and immunohistochemical analysis. Results indicate that the viability of SMSC in hydrogels treated with TGF-β1, TGF-β3 and BMP-2 remained high at culture time. The constructs formed cartilaginous tissue with the expression of chondrocytic genes (collagen type II, aggrecan, biglycan, SOX 9) and cartilaginous matrix (sulphated glycosaminoglycan and collagen) as early as 21 days in culture. Both TGF-β1 and TGF-β3 treated SMSC-laden hydrogels showed more chondrogenesis compared with BMP-2 treated SMSC-laden hydrogels. It demonstrates that injectable SMSC-laden gels, when treated with TGF-β1, TGF-β3 or BMP-2, are highly competent for in vitro engineered cartilage formation, which lays a foundation for their potential application in clinical cartilage repair.     

Improved Protocol for Chondrogenic Differentiation of Bone Marrow Derived Mesenchymal Stem Cells -Effect of PTHrP and FGF-2 on TGFβ1/BMP2-Induced Chondrocytes Hypertrophy

Growth factors have a pivotal role in chondrogenic differentiation of stem cells. The differential effects of known growth factors involved in the maintenance and homeostasis of cartilage tissue have been previously studied in vitro. However, there are few reported researches about the interactional effects of growth factors on chondrogenic differentiation of stem cells. The aim of this study is to examine the combined effects of four key growth factors on chondrogenic differentiation of mesenchymal stem cells (MSCs). Isolated and expanded rabbit bone marrow-derived MSCs underwent chondrogenic differentiation in a micromass cell culture system that used a combination of the following growth factors: transforming growth factor beta 1 (TGF-β1), bone morphogenetic protein 2 (BMP2), parathyroid hormone related protein (PTHrP), and fibroblast growth factor 2 (FGF2) according to a defined program. The chondrogenic differentiation program was analyzed by histochemistry methods, quantitative RT-PCR (qRT-PCR), and measurement of matrix deposition of sulfated glycosaminoglycan (sGAG) and collagen content at days 16, 23, and 30. The results showed that the short-term combination of TGF-β1 and BMP-2 increased sGAG and collagen content, Alkaline phosphates (ALP) activity, and type X collagen (COL X) expression. Application of either PTHrP or FGF2 simultaneously decreased TGF-β1/BMP-2 induced hypertrophy and chondrogenic markers (at least for FGF2). However, successive application of PTHrP and FGF2 dramatically maintained the synergistic effects of TGF-β1/BMP-2 on the chondrogenic differentiation potential of MSCs and decreased unwanted hypertrophic markers. This new method can be used effectively in chondrogenic differentiation programs.     

Chondrogenic potential of bone marrow- and adipose tissue-derived adult human mesenchymal stem cells

Regarding cartilage repair, tissue engineering is currently focusing on the use of adult mesenchymal stem cells (MSC) as an alternative to autologous chondrocytes. The potential of stem cells from various tissues to differentiate towards the chondrogenic phenotype has been investigated and it appears that the most common and studied sources are bone marrow (BM) and adipose tissue (AT) for historical and easy access reasons. In addition to three dimensional environment, the presence of member(s) of the transforming growth factor (TGF-β family and low oxygen tension have been reported to promote the in vitro differentiation of MSCs. Our work aimed at characterizing and comparing the degree of chondrogenic differentiation of MSCs isolated from BM and AT cultured in the same conditions. We also further aimed at and at determining whether hypoxia (2% oxygen) could affect the chondrogenic potential of AT-MSCs. Cells were first expanded in the presence of FGF-2, then harvested and centrifuged to allow formation of cell pellets, which were cultured in the presence of TGF-β3 and/or Bone Morphogenetic Protein-2 (BMP-2) and with 2 or 20% oxygen tension, for 24 days. Markers of the chondrocyte (COL2A1, AGC1, Sox9) and hypertrophic chondrocyte (COL10A1, MMP-13) were monitored by real-time PCR and/or by immunohistological staining. Our data show that BMP-2/TGF-β3 combination is the best culture condition to induce the chondrocyte phenotype in pellet cultures of BM and AT-MSCs. Particularly, a switch in the expression of the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of COL2A1 was observed. A parallel increase in gene expression of COL10A1 and MMP-13 was also recorded. However when AT-MSCs were cultured in hypoxia, the expression of markers of hypertrophic chondrocytes decreased when BMP-2/TGF-β3 were present in the medium. Thus it seems that hypoxia participates to the control of AT-MSCs chondrogenesis. Altogether, these cellular model systems will help us to investigate further the potential of different adult stem cells for cartilage engineering.     

滑膜间充质干细胞向透明软骨分化条件的体外研究

目的：研究TGFβ1、BMP-2、bFGF、IGF、BMP-7、ASA、Dex七种因素在滑膜间充质干细胞向透明软骨分化中起的作用。方法：酶消化法、有限稀释法获得滑膜间充质干细胞，并诱导其向脂肪细胞、软骨细胞和骨细胞分化。正交实验中纳入TGFβ1、BMP-2、bFGF、IGF、BMP-7、AsA、Dex七种因素。通过SPSS22.0统计软件设计L8(27)正交实验及表头，定义2水平条件。结果：滑膜间充质干细胞在成骨、成脂、成软骨分化后分别进行油红染色、碱性磷酸酶染色、甲苯胺蓝染色，染色结果均呈现为阳性。正交实验中直观观察、主体间方差分析显示TGFβ1作用最强。结论：滑膜间充质干细胞能够成功诱导向各种组织细胞分化，TGFβ1是七种因素中作用最明显的一种。     

Simultaneous regeneration of articular cartilage and subchondral bone in vivo using MSCs induced by a spatially controlled gene delivery system in bilayered integrated scaffolds

Engineering complex tissues is important but difficult to achieve in tissue regeneration. Osteochondral tissue engineering for the repair of osteochondral defect, involving simultaneous regeneration of bone and cartilage, has attracted considerable attention and also serves as an optimal model system for developing effective strategies aimed at regenerating complex tissues. In the present study, we formulated a bilayered gene-activated osteochondral scaffold consisting of plasmid TGF-β1-activated chitosan-gelatin scaffold for chondrogenic layer and plasmid BMP-2-activated hydroxyapatite/chitosan-gelatin scaffold for osteogenic layer. Mesenchymal stem cells seeded in each layer of the bilayered gene-?activated osteochondral scaffold showed significant cell proliferation, high expression of TGF-β1 protein and BMP-2 protein respectively. The results showed that spatially controlled and localized gene delivery system in the bilayered integrated scaffolds could induce the mesenchymal stem cells in different layers to differentiate into chondrocytes and osteoblasts in vitro, respectively, and simultaneously support the articular cartilage and subchondral bone regeneration in the rabbit knee ostochondral defect model. This study gives the evidence that multi-tissue regeneration through the combination of biomimetic and multi-phasic scaffold design, spatially controlled and localized gene delivery system and multi-lineage differentiation of a single stem cell population represents a promising strategy for facilitating the development of complex tissue or organ systems.     

In vivo ectopic chondrogenesis of BMSCs directed by mature chondrocytes

In vivo niche plays an important role in determining the fate of exogenously implanted stem cells. Due to the lack of a proper chondrogenic niche, stable ectopic chondrogenesis of mesenchymal stem cells (MSCs) in subcutaneous environments remains a great challenge. The clinical application of MSC-regenerated cartilage in repairing defects in subcutaneous cartilage such as nasal or auricular cartilage is thus severely limited. The creation of a chondrogenic niche in subcutaneous environments is the key to solving this problem. The current study demonstrates that bone marrow stromal cells (BMSCs) could form cartilage-like tissue in a subcutaneous environment when co-transplanted with articular chondrocytes, indicating that chondrocytes could create a chondrogenic niche to direct chondrogenesis of BMSCs. Then, a series of in vitro co-culture models revealed that it was the secretion of soluble factors by chondrocytes but not cell-cell contact that provided the chondrogenic signals. The subsequent studies further demonstrated that multiple factors currently used for chondroinduction (including TGF-β1, IGF-1 and BMP-2) were present in the supernatant of chondrocyte-engineered constructs. Furthermore, all of these factors were required for initiating chondrogenic differentiation and fulfilled their roles in a coordinated way. These results suggest that paracrine signaling of soluble chondrogenic factors provided by chondrocytes was an important mechanism in directing the in vivo ectopic chondrogenesis of BMSCs. The multiple co-culture systems established in this study provide new methods for directing committed differentiation of stem cells as well as new in vitro models for studying differentiation mechanism of stem cells determined by a tissue-specific niche.     

TGF-β3-induced chondrogenesis in co-cultures of chondrocytes and mesenchymal stem cells on biodegradable scaffolds

In this work, it was hypothesized that co-cultures of articular chondrocytes (ACs) and mesenchymal stem cells (MSCs) would exhibit enhanced sensitivity to chondrogenic stimuli, such as TGF-β3, and would require a reduced concentration of TGF-β3 to achieve an equivalent level of chondrogenesis compared to monocultures of each cell type. Furthermore, it was hypothesized that compared to monocultures, the chondrogenic phenotype of AC/MSC co-cultures would be more stable upon the removal of TGF-β3 from the culture medium. These hypotheses were investigated by culturing ACs and MSCs alone and in a 1:3 ratio on electrospun poly(?-caprolactone) scaffolds. All cell populations were cultured for two weeks with 0, 1, 3, or 10 ng/ml of TGF-β3. After two weeks growth factor supplementation was removed, and the constructs were cultured for two additional weeks. Cell proliferation, extracellular matrix production, and chondrogenic gene expression were evaluated after two and four weeks. The results demonstrated that co-cultures of ACs and MSCs require a reduced concentration and duration of TGF-β3 exposure to achieve an equivalent level of chondrogenesis compared to AC or MSC monocultures. Thus, the present work implicates that the promise of co-cultures for cartilage engineering is enhanced by their robust phenotype and heightened sensitivity to TGF-β3.     

Chondrogenesis of rabbit mesenchymal stem cells in fibrin/hyaluronan composite scaffold in vitro

Scaffold material is expected to play a crucial role in induction of chondrogenic differentiation of mesenchymal stem cells (MSCs) for cartilage tissue engineering. Here we demonstrated the feasibility of a fibrin/hyaluronan (HA) composite hydrogel as a potent scaffold for support of chondrogenesis of rabbit MSCs (rMSCs). rMSCs were prepared in three-dimensional cultures of pellet, alginate layer, and fibrin/HA gel. Specimens in each group were cultured in chondrogenic defined media for 4 weeks in the absence or presence of transforming growth factor β1 (TGF-β1) treatment. Viability of rMSCs was somewhat reduced until 4 weeks, which was less significant in fibrin/HA gels than in the alginate layer (*p?相似文献   

In vitro cartilage tissue engineering using adipose-derived extracellular matrix scaffolds seeded with adipose-derived stem cells

Extracellular matrix (ECM) secreted from the resident cell of tissue is an ideal biomaterial evolved by nature. Cartilage is also built from well-organized ECM components in a gel-like structure with a high collagen and proteoglycan content. Here, we explored cartilage tissue engineering using ECM scaffolds seeded with stem cells. Both scaffolds and stem cells were isolated from human adipose tissue, which is abundant and easily harvested in the human body. The human ECM scaffolds contained various endogenous bioactive factors, including transforming growth factor-beta1 (TGF-β1, 8782±4989?pg/g, dry ECM), insulin growth factor-1 (13319±1388?pg/g, dry ECM), basic fibroblast growth factor (82373±9572?pg/g, dry ECM), and vascular endothelial growth factor (25647±2749?pg/g, dry ECM). A composite of ECM and stem cells was prepared and cultured in chondrogenic medium (with 10?ng/mL TGF-β1 or not) for 45 days. The volumes and weights of the composites increased during culture and the surface gradually became smooth. Cell viability remained high throughout the 45 days of in vitro culture. Composites showed the formation of cartilage-like tissue with the synthesis of cartilage-specific proteins such as collagen and glycosaminoglycan. Important chondrogenic markers were expressed including Sox-9, aggrecan, and collagen type II and XI. These results demonstrate that a cell/ECM composite containing endogenous bioactive factors could provide biochemical cues for the promotion of cartilage formation.     

Expression of chondrogenic genes by undifferentiated vs. differentiated human mesenchymal stem cells using array technology

This study investigated the gene expression profile of human mesenchymal stem cells seeded in collagen sponge for 28 days in three different mediums: (1) basal medium as control containing ITS alone, (2) ITS+TGF-β1 alone or (3) ITS 1% supplemented sequentially by TGF-β1 (D3-D14) followed by BMP-2 (D15-D28). Differential expression of 84 genes implicated in chondrogenic and osteogenic differentiation of MSCs was analyzed at D28 by real-time RT-PCR array technology. TGF-β1 alone down-regulated two genes, CD36 and cathepsin K. Sixteen genes were significantly up-regulated, notably type 2 and type 10 collagens, COMP and Sox9. The sequential combination of TGF-β1 and BMP-2 produced a similar profile with prominent expression of type 2 collagen and the alkaline phosphatase gene. Interestingly, in this in vitro condition, RUNX2 was not up-regulated, suggesting that the sequential combination of TGF-β1/BMP2 enhances the hypertrophic chondrogenic profile without turning towards the osteoblastic pathway.     

Adenovirus-mediated expression of growth and differentiation factor-5 promotes chondrogenesis of adipose stem cells

The repair of articular cartilage injuries is impeded by the avascular and non-innervated nature of cartilage. Transplantation of autologous chondrocytes has a limited ability to augment the repair process due to the highly differentiated state of chondrocytes and the risks of donor-site morbidity. Mesenchymal stem cells can undergo chondrogenesis in the presence of growth factors for cartilage defect repair. Growth and differentiation factor-5 (GDF5) plays an important role in chondrogenesis. In this study, we examined the effects of GDF5 on chondrogenesis of adipose-derived stem cells (ADSCs) and evaluate the chondrogenic potentials of GDF5 genetically engineered ADSCs using an in vitro pellet culture model. Rat ADSCs were grown as pellet cultures and treated with chondrogenic media (CM). Induction of GDF5 by an adenovirus (Ad-GDF5) was compared with exogenous supplementation of GDF5 (100 ng/ml) and transforming growth factor-β (TGF-β1; 10 ng/ml). The ADSCs underwent chondrogenic differentiation in response to GDF5 exposure as demonstrated by production of proteoglycan, and up-regulation of collagen II and aggrecan at the protein and mRNA level. The chondrogenic potential of a one-time infection with Ad-GDF5 was weaker than exogenous GDF5, but equal to that of TGF-β1. Stimulation with growth factors or CM alone induced transient expression of the mRNA for collagen X, indicating a need for optimization of the CM. Our findings indicate that GDF5 is a potent inducer of chondrogenesis in ADSCs, and that ADSCs genetically engineered to express prochondrogenic growth factors, such as GDF5, may be a promising therapeutic cell source for cartilage tissue engineering.     

Effect of dynamic loading on MSCs chondrogenic differentiation in 3-D alginate culture

Mesenchymal stem cells (MSCs) are regarded as a potential autologous source for cartilage repair, because they can differentiate into chondrocytes by transforming growth factor-beta (TGF-β) treatment under the 3-dimensional (3-D) culture condition. In addition to these molecular and biochemical methods, the mechanical regulation of differentiation and matrix formation by MSCs is only starting to be considered. Recently, mechanical loading has been shown to induce chondrogenesis of MSCs in vitro. In this study, we investigated the effects of a calibrated agitation on the chondrogenesis of human bone MSCs (MSCs) in a 3-D alginate culture (day 28) and on the maintenance of chondrogenic phenotypes. Biomechanical stimulation of MSCs increased: (i) types 1 and 2 collagen formation; (ii) the expression of chondrogenic markers such as COMP and SOX9; and (iii) the capacity to maintain the chondrogenic phenotypes. Notably, these effects were shown without TGF-β treatment. These results suggest that a mechanical stimulation could be an efficient method to induce chondrogenic differentiation of MSCs in vitro for cartilage tissue engineering in a 3-D environment. Additionally, it appears that MSCs and chondrocyte responses to mechanical stimulation are not identical.     

In situ chondrogenic differentiation of human adipose tissue-derived stem cells in a TGF-β1 loaded fibrin–poly(lactide-caprolactone) nanoparticulate complex

When conducting cartilage tissue engineering with stem cells, it is well known that chemical and physical stimulations are very important for the induction and maintenance of chondrogenesis. In this study, we induced chondrogenic differentiation of human adipose tissue-derived stem cells (hASCs) in situ by effective stimulation via the continuous controlled release of TGF-β1 from a heparin-functionalized nanoparticle–fibrin–poly(lactide-co-caprolactone) (PLCL) complex. PLCL scaffolds were fabricated with 85% porosity and 300–500 μm pore size by a gel-pressing method. Heparin-functionalized nanoparticles were prepared by a solvent-diffusion method, composed of poly(lactide-co-glycolide) (PLGA), Pluronic F-127, and heparin, and then TGF-β1 was loaded to the nanoparticles. A mixture of hASCs, fibrin gels and TGF-β1 loaded nanoparticles was then seeded onto PLCL scaffolds and cultured in vitro, after which they were subcutaneously implanted into nude mice for up to five weeks. The results of in vitro and in vivo studies revealed that chondrogenic differentiation of the hASCs on the complex was induced and sustained by continuous stimulation by TGF-β1 from the heparin-functionalized nanoparticles. In addition, there was no significant difference between the predifferentiation condition prior to incubation in chondrogenic medium and the proliferation condition, which suggests that in situ chondrogenic differentiation of hASCs was induced by the TGF-β1 loaded nanoparticles. Consequently, the hybridization of fibrin and PLCL scaffolds for three-dimensional spatial organization of cells and the effective delivery of TGF-β1 using heparin-functionalized nanoparticles can induce hASCs to differentiate to a chondrogenic lineage and maintain their phenotypes.     

Mechano growth factor (MGF) and transforming growth factor (TGF)-β3 functionalized silk scaffolds enhance articular hyaline cartilage regeneration in rabbit model

Damaged cartilage has poor self-healing ability and usually progresses to scar or fibrocartilaginous tissue, and finally degenerates to osteoarthritis (OA). Here we demonstrated that one of alternative isoforms of IGF-1, mechano growth factor (MGF) acted synergistically with transforming growth factor β3 (TGF-β3) embedded in silk fibroin scaffolds to induce chemotactic homing and chondrogenic differentiation of mesenchymal stem cells (MSCs). Combination of MGF and TGF-β3 significantly increased cell recruitment up to 1.8 times and 2 times higher than TGF-β3 did in vitro and in vivo. Moreover, MGF increased Collagen II and aggrecan secretion of TGF-β3 induced hMSCs chondrogenesis, but decreased Collagen I in vitro. Silk fibroin (SF) scaffolds have been widely used for tissue engineering, and we showed that methanol treated pured SF scaffolds were porous, similar to compressive module of native cartilage, slow degradation rate and excellent drug released curves. At 7days after subcutaneous implantation, TGF-β3 and MGF functionalized silk fibroin scaffolds (STM) recruited more CD29+/CD44 + cells (P < 0.05). Similarly, more cartilage-like extracellular matrix and less fibrillar collagen were detected in STM scaffolds than that in TGF-β3 modified scaffolds (ST) at 2 months after subcutaneous implantation. When implanted into articular joints in a rabbit osteochondral defect model, STM scaffolds showed the best integration into host tissues, similar architecture and collagen organization to native hyaline cartilage, as evidenced by immunostaining of aggrecan, collagen II and collagen I, as well as Safranin O and Masson's trichrome staining, and histological evalution based on the modified O'Driscoll histological scoring system (P < 0.05), indicating that MGF and TGF-β3 might be a better candidate for cartilage regeneration. This study demonstrated that TGF-β3 and MGF functionalized silk fibroin scaffolds enhanced endogenous stem cell recruitment and facilitated in situ articular cartilage regeneration, thus providing a novel strategy for cartilage repair.     

Differentiation of adult human mesenchymal stem cells: chondrogenic effect of BMP-2

Articular cartilage is essential for the motion of the skeleton. However, this tissue is unable to spontaneously repair once injured, since it is avascular and aneural. Numerous repair strategies are developed, but they do not lead to a functional tissue and research into cartilage repair focuses now on tissue engineering technics. Adult mesenchymal stem cells (MSC), present in various tissues, have the potential to differentiate into chondrocytes in vitro in response to specific growth factors. The members of the transforming growth factor beta, among them the bone morphogenetic protein (BMP)-2, appear very promising inducers in this context. BMP-2 favours chondrogenic expression, in particular expression of type IIB collagen, the cartilage-specific isoform of this collagen. Therefore, collagen type IIB is a good indicator of the differentiation state of MSC. However, since BMP-2 has also osteogenic properties, it is critical to differentially control chondrogenic and osteogenic properties of BMP-2 when used with MSC. Strategies for this control are presented in this review. Most likely, this is the combination of growth factors such as BMP-2 with biomaterials that will lead to the successful use of MSC for cartilage repair.     

Generation of osteochondral tissue constructs with chondrogenically and osteogenically predifferentiated mesenchymal stem cells encapsulated in bilayered hydrogels

This study investigated the ability of chondrogenic and osteogenic predifferentiation of mesenchymal stem cells (MSCs) to play a role in the development of osteochondral tissue constructs using injectable bilayered oligo(poly(ethylene glycol) fumarate) (OPF) hydrogel composites. We hypothesized that the combinatorial approach of encapsulating cell populations of both chondrogenic and osteogenic lineages in a spatially controlled manner within bilayered constructs would enable these cells to maintain their respective phenotypes via the exchange of biochemical factors even without the influence of external growth factors. During monolayer expansion prior to hydrogel encapsulation, it was found that 7 (CG7) and 14 (CG14) days of MSC exposure to TGF-β3 allowed for the generation of distinct cell populations with corresponding chondrogenic maturities as indicated by increasing aggrecan and type II collagen/type I collagen expression. Chondrogenic and osteogenic cells were then encapsulated within their respective (chondral/subchondral) layers in bilayered hydrogel composites to include four experimental groups. Encapsulated CG7 cells within the chondral layer exhibited enhanced chondrogenic phenotype when compared to other cell populations based on stronger type II collagen and aggrecan gene expression and higher glycosaminoglycan-to-hydroxyproline ratios. Osteogenic cells that were co-cultured with chondrogenic cells (in the chondral layer) showed higher cellularity over time, suggesting that chondrogenic cells stimulated the proliferation of osteogenic cells. Groups with osteogenic cells displayed mineralization in the subchondral layer, confirming the effect of osteogenic predifferentiation. In summary, it was found that MSCs that underwent 7 days, but not 14 days, of chondrogenic predifferentiation most closely resembled the phenotype of native hyaline cartilage when combined with osteogenic cells in a bilayered OPF hydrogel composite, indicating that the duration of chondrogenic preconditioning is an important factor to control. Furthermore, the respective chondrogenic and osteogenic phenotypes were maintained for 28 days in vitro without the need for external growth factors, demonstrating the exciting potential of this novel strategy for the generation of osteochondral tissue constructs for cartilage engineering applications.     

Chondrogenic and osteogenic differentiations of human bone marrow-derived mesenchymal stem cells on a nanofibrous scaffold with designed pore network

The utilization of 3D scaffolds and stem cells is a promising approach to solve the problem of bone and cartilage tissue shortage and to construct osteochondral (cartilage/bone composite) tissues. In this study, 3D highly porous nanofibrous (NF) poly(l-lactic acid) (PLLA) scaffolds fabricated using a phase separation technique were seeded with multi-potent human bone marrow-derived mesenchymal stem cells (hMSCs) and the constructs were induced along osteogenic and chondrogenic development routes in vitro. Histological analysis and calcium content quantification showed that NF scaffolds supported in vitro bone differentiation. SEM observation showed an altered shape for cells cultured on an NF matrix compared with those on smooth films. Consistent with the morphological change, the gene expression of early chondrogenic commitment marker Sox-9 was enhanced on the NF matrix. NF scaffolds were then used to support long-term in vitro 3D cartilaginous development. It was found that in the presence of TGF-β1, cartilage tissue developed on PLLA NF scaffolds, with the cartilage-specific gene expressed, glycosaminoglycan and type II collagen accumulated, and typical cartilage morphology formed. These findings suggest that NF scaffolds can support both bone and cartilage development and are excellent candidate scaffolds for osteochondral defect repair.     

Dental mesenchymal stem cells encapsulated in an alginate hydrogel co-delivery microencapsulation system for cartilage regeneration

Dental-derived mesenchymal stem cells (MSCs) are promising candidates for cartilage regeneration, with a high capacity for chondrogenic differentiation. This property helps make dental MSCs an advantageous therapeutic option compared to current treatment modalities. The MSC delivery vehicle is the principal determinant for the success of MSC-mediated cartilage regeneration therapies. The objectives of this study were to: (1) develop a novel co-delivery system based on TGF-β1 loaded RGD-coupled alginate microspheres encapsulating periodontal ligament stem cells (PDLSCs) or gingival mesenchymal stem cells (GMSCs); and (2) investigate dental MSC viability and chondrogenic differentiation in alginate microspheres. The results revealed the sustained release of TGF-β1 from the alginate microspheres. After 4 weeks of chondrogenic differentiation in vitro, PDLSCs and GMSCs as well as human bone marrow mesenchymal stem cells (hBMMSCs) (as positive control) revealed chondrogenic gene expression markers (Col II and Sox-9) via qPCR, as well as matrix positively stained by Toluidine Blue and Safranin-O. In animal studies, ectopic cartilage tissue regeneration was observed inside and around the transplanted microspheres, confirmed by histochemical and immunofluorescent staining. Interestingly, PDLSCs showed more chondrogenesis than GMSCs and hBMMSCs (p < 0.05). Taken together, these results suggest that RGD-modified alginate microencapsulating dental MSCs make a promising candidate for cartilage regeneration. Our results highlight the vital role played by the microenvironment, as well as value of presenting inductive signals for viability and differentiation of MSCs.     

Controlled release of transforming growth factor-β3 from cartilage-extra-cellular-matrix-derived scaffolds to promote chondrogenesis of human-joint-tissue-derived stem cells

The objective of this study was to develop a scaffold derived from cartilaginous extracellular matrix (ECM) that could be used as a growth factor delivery system to promote chondrogenesis of stem cells. Dehydrothermal crosslinked scaffolds were fabricated using a slurry of homogenized porcine articular cartilage, which was then seeded with human infrapatellar-fat-pad-derived stem cells (FPSCs). It was found that these ECM-derived scaffolds promoted superior chondrogenesis of FPSCs when the constructs were additionally stimulated with transforming growth factor (TGF)-β3. Cell-mediated contraction of the scaffold was observed, which could be limited by the additional use of 1-ethyl-3-3dimethyl aminopropyl carbodiimide (EDAC) crosslinking without suppressing cartilage-specific matrix accumulation within the construct. To further validate the utility of the ECM-derived scaffold, we next compared its chondro-permissive properties to a biomimetic collagen–hyaluronic acid (HA) scaffold optimized for cartilage tissue engineering (TE) applications. The cartilage-ECM-derived scaffold supported at least comparable chondrogenesis to the collagen–HA scaffold, underwent less contraction and retained a greater proportion of synthesized sulfated glycosaminoglycans. Having developed a promising scaffold for TE, with superior chondrogenesis observed in the presence of exogenously supplied TGF-β3, the final phase of the study explored whether this scaffold could be used as a TGF-β3 delivery system to promote chondrogenesis of FPSCs. It was found that the majority of TGF-β3 that was loaded onto the scaffold was released in a controlled manner over the first 10 days of culture, with comparable long-term chondrogenesis observed in these TGF-β3-loaded constructs compared to scaffolds where the TGF-β3 was continuously added to the media. The results of this study support the use of cartilage-ECM-derived scaffolds as a growth factor delivery system for use in articular cartilage regeneration.