Xenogeneic chondrocytes promote stable subcutaneous chondrogenesis of bone marrow-derived stromal cells

The local microenvironment may change the ultimate fate of engineered cartilage differentiated from bone marrow stromal cells (BMSCs) after subcutaneous implantation. Chondrogenically differentiated BMSCs directed by growth factors or low-intensity ultrasound are apt to fibrose or vascularize in the subcutaneous environment, while BMSCs implanted in articular cartilage defects can form stable cartilage. We hypothesized that chondrocytes would provide an ideal chondrogenic environment, and thus promote the maintenance of the chondrocytic phenotype in ectopia. To test this hypothesis, we developed a new method to promote chondrocyte development from BMSCs in a chondrogenic environment produced by xenogeneic chondrocytes and compared the subcutaneous chondrogenesis of BMSCs mediated by xenogeneic chondrocytes with that produced by growth factors. These results indicate that subcutaneous chondrogenesis of BMSCs directed by xenogeneic chondrocytes is more effective than that induced by growth factors. BMSCs induced by xenogeneic chondrocytes formed relatively mature cartilage before or after implantation, following 4 weeks of culture, which reduced the induction time in?vitro and led to maintenance of a stable cartilage phenotype after subcutaneous implantation.     

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.     

Enhanced MSC chondrogenesis following delivery of TGF-β3 from alginate microspheres within hyaluronic acid hydrogels in vitro and in vivo

Mesenchymal stem cells (MSCs) are being recognized as a viable cell source for cartilage repair and members of the transforming growth factor-beta (TGF-β) superfamily are a key mediator of MSC chondrogenesis. While TGF-β mediated MSC chondrogenesis is well established in in vitro pellet or hydrogel cultures, clinical translation will require effective delivery of TGF-βs in vivo. Here, we investigated the co-encapsulation of TGF-β3 containing alginate microspheres with human MSCs in hyaluronic acid (HA) hydrogels towards the development of implantable constructs for cartilage repair. TGF-β3 encapsulated in alginate microspheres with nanofilm coatings showed significantly reduced initial burst release compared to uncoated microspheres, with release times extending up to 6 days. HA hydrogel constructs seeded with MSCs and TGF-β3 containing microspheres developed comparable mechanical properties and cartilage matrix content compared to constructs supplemented with TGF-β3 continuously in culture media, whereas constructs with TGF-β3 directly encapsulated in the gels without microspheres had inferior properties. When implanted subcutaneously in nude mice, constructs containing TGF-β3 microspheres resulted in superior cartilage matrix formation when compared to groups without TGF-β3 or with TGF-β3 added directly to the gel. However, calcification was observed in implanted constructs after 8 weeks of subcutaneous implantation. To prevent this, the co-delivery of parathyroid hormone-related protein (PTHrP) with TGF-β3 in alginate microspheres was pursued, resulting in partially reduced calcification. This study demonstrates that the controlled local delivery of TGF-β3 is essential to neocartilage formation by MSCs and that further optimization is needed to avert the differentiation of chondrogenically induced MSCs towards a hypertrophic phenotype.     

Regeneration of human-ear-shaped cartilage by co-culturing human microtia chondrocytes with BMSCs

Previously, we had addressed the issues of shape control/maintenance of in vitro engineered human-ear-shaped cartilage. Thus, lack of applicable cell source had become a major concern that blocks clinical translation of this technology. Autologous microtia chondrocytes (MCs) and bone marrow stromal cells (BMSCs) were both promising chondrogenic cells that did not involve obvious donor site morbidity. However, limited cell availability of MCs and ectopic ossification of chondrogenically induced BMSCs in subcutaneous environment greatly restricted their applications in external ear reconstruction. The current study demonstrated that MCs possessed strong proliferation ability but accompanied with rapid loss of chondrogenic ability during passage, indicating a poor feasibility to engineer the entire ear using expanded MCs. Fortunately, the co-transplantation results of MCs and BMSCs (25% MCs and 75% BMSCs) demonstrated a strong chondroinductive ability of MCs to promote stable ectopic chondrogenesis of BMSCs in subcutaneous environment. Moreover, cell labeling demonstrated that BMSCs could transform into chondrocyte-like cells under the chondrogenic niche provided by co-cultured MCs. Most importantly, a human-ear-shaped cartilaginous tissue with delicate structure and proper elasticity was successfully constructed by seeding the mixed cells (MCs and BMSCs) into the pre-shaped biodegradable ear-scaffold followed by 12 weeks of subcutaneous implantation in nude mouse. These results may provide a promising strategy to construct stable ectopic cartilage with MCs and stem cells (BMSCs) for autologous external ear reconstruction.     

Fluorescently labeled mesenchymal stem cells (MSCs) maintain multilineage potential and can be detected following implantation into articular cartilage defects.

Several studies have reported enhanced repair of damaged cartilage following implantation of mesenchymal stem cells (MSCs) into full-thickness cartilage defects suggesting that the cells in the repair tissue were derived from the implant. However, it cannot be excluded that the enhanced tissue repair is derived from host cells recruited to the defect in response to the implant, rather than the re-population of the tissue by the implanted MSCs. Our objective was to study the short-term fate of fluorescently labeled MSCs after implantation into full-thickness cartilage defects in vivo. The fluorescent dye used in our studies did not affect MSC viability or their ability to undergo osteogenic and chondrogenic differentiation in vitro. MSC gelatin constructs were implanted into full-thickness cartilage defects in goats. These cells retained the dye and were detectable by histology and flow cytometry. At intervals spanning 2 weeks post-implantation we observed gradual loss of implanted cells in the defect as well as fragments of gelatin sponge containing labeled MSCs in deep marrow spaces indicating fragmentation, dislodgement and passive migration. Fluorescent labeling enabled us to determine whether the implanted cells were lost during early time points after implantation as well as their spatial orientation throughout the defect. By determining the fate of implanted cells, new biomaterials could be engineered to correct undesirable characteristics. Testing of new biomaterials in short-term in vivo models would provide faster optimization for cell retention needed for successful, long-term cartilage regeneration.     

Hypoxia impedes hypertrophic chondrogenesis of human multipotent stromal cells

Within the field of bone tissue engineering, the endochondral approach to forming bone substitutes represents a novel concept, where cartilage will undergo hypertrophic differentiation before its conversion into bone. For this purpose, clinically relevant multipotent stromal cells (MSCs), MSCs, can be differentiated into the chondrogenic lineage before stimulating hypertrophy. Controversy exists in literature on the oxygen tensions naturally present during this transition in, for example, the growth plate. Therefore, the present study focused on the effects of different oxygen tensions on the progression of the hypertrophic differentiation of MSCs. Bone marrow-derived MSCs of four human donors were expanded, and differentiation was induced in aggregate cultures. Normoxic (20% oxygen) and hypoxic (5%) conditions were imposed on the cultures in chondrogenic or hypertrophic differentiation media. After 4 weeks, the cultures were histologically examined and by real-time polymerase chain reaction. Morphological assessment showed the chondrogenic differentiation of cultures from all donors under normoxic chondrogenic conditions. In addition, hypertrophic differentiation was observed in cultures derived from all but one donor. The deposition of collagen type X was evidenced in both chondrogenically and hypertrophically stimulated cultures. However, mineralization was exclusively observed in hypertrophically stimulated, normoxic cultures. Overall, the progression of hypertrophy was delayed in hypoxic compared with normoxic groups. The observed delay was supported by the gene expression patterns, especially showing the up-regulation of the late hypertrophic markers osteopontin and osteocalcin under normoxic hypertrophic conditions. Concluding, normoxic conditions are more beneficial for hypertrophic differentiation of MSCs than are hypoxic conditions, as long as the MSCs possess hypertrophic potential. This finding has implications for cartilage tissue engineering as well as for endochondral bone tissue engineering, as these approaches deal with, respectively, the inhibition or enhancement of hypertrophic chondrogenesis.     

Ectopic neocartilage formation from predifferentiated human adipose derived stem cells induced by adenoviral-mediated transfer of hTGF beta2

Chondrogenic potential of human adipose derived stem cells (hASCs) makes them a possible source of seeding cells for cartilage tissue engineering. In this study, chondrogenic differentiation of hASCs induced by transduction with replication-deficient adenovirus carrying human transforming growth factor beta2 (Ad5-hTGF beta2) was demonstrated by RT-PCR, immunohistochemistry staining, biochemical and western blot analysis. To evaluate if the in vitro differentiated hASCs could keep their chondrocytic phenotype and produce neo-cartilage in vivo, predifferentiated hASCs were seeded in different scaffolds and implanted in subcutaneous pockets on the dorsum of nude mice. After 4 and 12 weeks culture in vivo, specimens were harvested and examined by histological and immunohistochemical analysis, cartilage-like tissue formation was only found in alginate gel and PLGA/alginate compound groups, in PLGA group, fibrous tissues and angiogenesis ingrowth were observed. These findings demonstrated that adenovirus-mediated hTGF beta2 gene transfer could induce hASCs into a chondrogenic lineage in vitro, however, this predifferentiation did not guarantee ectopic cartilage formation in vivo unless appropriate three-dimensional scaffolds were used as the cell carry vehicles.     

Engineering osteochondral constructs through spatial regulation of endochondral ossification

Chondrogenically primed bone marrow-derived mesenchymal stem cells (MSCs) have been shown to become hypertrophic and undergo endochondral ossification when implanted in vivo. Modulating this endochondral phenotype may be an attractive approach to engineering the osseous phase of an osteochondral implant. The objective of this study was to engineer an osteochondral tissue by promoting endochondral ossification in one layer of a bilayered construct and stable cartilage in the other. The top half of bilayered agarose hydrogels were seeded with culture expanded chondrocytes (termed the chondral layer) and the bottom half of the bilayered agarose hydrogels with MSCs (termed the osseous layer). Constructs were cultured in chondrogenic medium for 21 days and thereafter were either maintained in chondrogenic medium, transferred to hypertrophic medium, or implanted subcutaneously into nude mice. This structured chondrogenic bilayered co-culture was found to enhance chondrogenesis in the chondral layer, appearing to help re-establish the chondrogenic phenotype that is lost in chondrocytes during monolayer expansion. Furthermore, the bilayered co-culture appeared to suppress hypertrophy and mineralization in the osseous layer. The addition of hypertrophic factors to the media was found to induce mineralization of the osseous layer in vitro. A similar result was observed in vivo where endochondral ossification was restricted to the osseous layer of the construct, leading to the development of an osteochondral tissue. This novel approach represents a potential new treatment strategy for the repair of osteochondral defects.     

Smad signaling determines chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells: inhibition of Smad1/5/8P prevents terminal differentiation and calcification

The aim of this study was to investigate the roles of Smad2/3 and Smad1/5/8 phosphorylation in transforming growth factor-beta-induced chondrogenic differentiation of bone-marrow-derived mesenchymal stem cells (BMSCs) to assess whether specific targeting of different Smad signaling pathways offers possibilities to prevent terminal differentiation and mineralization of chondrogenically differentiated BMSCs. Terminally differentiated chondrocytes produced in vitro by chondrogenic differentiation of BMSCs or studied ex vivo during murine embryonic limb formation stained positive for both Smad2/3P and Smad1/5/8P. Hyaline-like cartilage produced in vitro by articular chondrocytes or studied in ex vivo articular cartilage samples that lacked expression for matrix metalloproteinase 13 and collagen X only expressed Smad2/3P. When either Smad2/3 or Smad1/5/8 phosphorylation was blocked in BMSC culture by addition of SB-505124 or dorsomorphin throughout culture, no collagen II expression was observed, indicating that both pathways are involved in early chondrogenesis. Distinct functions for these pathways were demonstrated when Smad signaling was blocked after the onset of chondrogenesis. Blocking Smad2/3P after the onset of chondrogenesis resulted in a halt in collagen II production. On the other hand, blocking Smad1/5/8P during this time period resulted in decreased expression of matrix metalloproteinase 13, collagen X, and alkaline phosphatase while allowing collagen II production. Moreover, blocking Smad1/5/8P prevented mineralization. This indicates that while Smad2/3P is important for continuation of collagen II deposition, Smad1/5/8 phosphorylation is associated with terminal differentiation and mineralization.     

Species variability in the differentiation potential of in vitro-expanded articular chondrocytes restricts predictive studies on cartilage repair using animal models

Autologous chondrocyte implantation is currently applied in clinics as an innovative tool for articular cartilage repair. Animal models have been and still are being used to validate and further improve the technique. However, in various species, the outcome varies from hyaline-like cartilage to fibrocartilage. This may be due partly to the spontaneous dedifferentiation of chondrocytes once cultured in vitro. Here we assessed whether the extent of dedifferentiation varies between species and we hypothesized that the level of chondrocyte phenotype stability during expansion may contribute to the maintenance of their chondrogenic commitment and redifferentiation potential. Condyle chondrocytes were harvested from sheep, dog, and human, and expanded for 1, 6, or 12 cell duplications. At each interval, cell phenotype was monitored (morphology and biosynthesis of cartilage markers) and redifferentiation was assessed by an in vitro assay of chondrogenesis in micromass pellet and an in vivo assay of ectopic cartilage formation in immunodeficient mice. Results indicate that, during culture, the sheep chondrocyte phenotype is maintained better than that of human chondrocytes, which in turn dedifferentiate to a lesser extent than dog chondrocytes Accordingly, after expansion, sheep chondrocytes spontaneously reform hyaline-like cartilage; human chondrocytes redifferentiate only under stimulation with chondrogenic inducers whereas, after a few passages, dog chondrocytes lose any capacity to redifferentiate regardless of the presence of inducers. Thus, conditions allowing cartilage formation in one species are not necessarily transposable to other species. Therefore, results with animal models should be cautiously applied to humans. In addition, for tissue-engineering purposes, the number of cell duplications must be, for each species, carefully monitored to remain in the range of amplification allowing redifferentiation and chondrogenesis.     

Temporal activation of β-catenin signaling in the chondrogenic process of mesenchymal stem cells affects the phenotype of the cartilage generated

Adult mesenchymal stem cells (MSCs) are an attractive cell source for cartilage tissue engineering. In vitro predifferentiation of MSCs has been explored as a means to enhance MSC-based articular cartilage repair. However, there remain challenges to control and prevent the premature progression of MSC-derived chondrocytes to the hypertrophy. This study investigated the temporal effect of transforming growth factor (TGF)-β and β-catenin signaling co-activation during MSC chondrogenic differentiation and evaluated the influence of these predifferentiation conditions to subsequent phenotypic development of the cartilage. MSCs were differentiated in chondrogenic medium that contained either TGFβ alone, TGFβ with transient β-catenin coactivation, or TGFβ with continuous β-catenin coactivation. After in vitro differentiation, the pellets were transplanted into SCID mice. Both coactivation protocols resulted in the enhancement of chondrogenic differentiation of MSCs. Compared with TGFβ activation, transient coactivation of TGFβ-induction with β-catenin activation resulted in heightened hypertrophy and formed highly ossified tissues with marrow-like hematopoietic tissue in vivo. The continuous coactivation of the 2 signaling pathways, however, resulted in inhibition of progression to hypertrophy, marked by the suppression of type X collagen, Runx2, and alkaline phosphatase expression, and did not result in ossified tissue in vivo. Chondrocytes of the continuous co-activation samples secreted significantly more parathyroid hormone-related protein (PTHrP) and expressed cyclin D1. Our results suggest that temporal co-activation of the TGFβ signaling pathway with β-catenin can yield cartilage of different phenotype, represents a potential MSC predifferentiation protocol before clinical implantation, and has potential applications for the engineering of cartilage tissue.     

Combined chondrocyte-copolymer implantation with slow release of basic fibroblast growth factor for tissue engineering an auricular cartilage construct

Basic fibroblast growth factor (b-FGF) may have a role in tissue-engineered chondrogenesis. However, when applied in solution, b-FGF rapidly diffuses from the implant site. In another approach for tissue engineering, poly-lactide-based copolymers have shown promise as scaffolds for chondrocytes used to tissue engineer auricular cartilage in the shape of an ear. This study evaluated the effectiveness of b-FGF impregnated in gelatin microspheres to achieve slow growth factor release for augmenting the in vivo chondrogenic response. Whereas 125I-labeled b-FGF injected in solution showed rapid in vivo clearance from the injection site (only 3% residual after 24 h), when incorporated into gelatin microspheres, 44% and 18% of the b-FGF remained at 3 and 14 days, respectively. Canine chondrocytes were isolated and grown in vitro onto ear-shaped poly-lactide/caprolactone copolymers for 1 week, then implanted into the dorsal subcutaneous tissue of nude mice; implants contained b-FGF either in free solution or in gelatin microspheres. A third group underwent preinjection of b-FGF in gelatin microspheres 4 days before chondrocyte-copolymer implantation. The implants with b-FGF-incorporated microspheres showed the greatest chondrogenic characteristics at 5 and 10 weeks postoperatively: good shape and biomechanical trait retention, strong (histologic) metachromasia, rich vascularization of surrounding tissues, and increased gene expression for type II collagen (cartilage marker) and factor VIII-related antigen (vascular marker). In the case of implant site preadministration with b-FGF-impregnated microspheres, the implant architecture was not maintained as well, and reduced vascularization and metachromasia was also apparent. In conclusion, these findings indicate that a sustained release of b-FGF augments neovascularization and chondrogenesis in a tissue-engineered cartilage construct.     

Effects of hypoxias and scaffold architecture on rabbit mesenchymal stem cell differentiation towards a nucleus pulposus-like phenotype

Nucleus pulposus grafts are needed for patients requiring replacement of their degenerated intervertebral discs. Bone marrow-derived mesenchymal stem cells (MSCs) are a potential autologous stem cell source for the nucleus pulposus regeneration. One of the key issues of constructing functional nucleus pulposus using MSCs, however, is to differentiate MSCs into nucleus pulposus phenotype in?vitro and to maintain their phenotypic stability in?vivo. In this study, three-dimensional (3D) nanofibrous poly(l-lactide) (PLLA) scaffolds were seeded with multi-potent rabbit MSCs and the constructs were induced along nucleus pulposus development routes in a hypoxia chamber (2% O(2)) in the presence of TGF-β1. It was found that nanofibrous scaffold could support the differentiation of rabbit MSCs towards a nucleus pulposus-like phenotype in?vitro, as evidenced by upregulated expression of a few important nucleus pulposus-associated genes (aggrecan, type II collagen and Sox-9), abundant deposition of extracellular matrix (glycosaminoglycan (GAG) and type II collagen), and the continuous expression of the nucleus pulposus-specific marker, hypoxia-inducible factor (HIF)-1α. The subcutaneous implantation results confirmed that hypoxic induction before implantation could help the constructs to retain their phenotype and resist calcification in?vivo. Therefore, the above data showed the promise of using 3D nanofibrous scaffolds in combination with TGF-β1 and hypoxic induction to regenerate functional nucleus pulposus grafts for intervertebral disc replacement.     

Ectopic osteochondral formation of biomimetic porous PVA-n-HA/PA6 bilayered scaffold and BMSCs construct in rabbit

In this work, the novel poly vinyl alcohol/gelatin-nano-hydroxyapatite/polyamide6 (PVA-n-HA/PA6) bilayered scaffold with biomimetic properties for articular cartilage and subchondral bone is developed. Furthermore, when these osteochondral scaffolds were seeded with induced bone mesenchymal stem cells (BMSCs) and implanted at ectopic sites, showed the potential for an engineered cartilage tissue and the corresponding subchondral bone. BMSCs were expanded in vitro and induced to chondrogenic or osteogenic potential by culturing in suitable media for 14 days. Subsequently, these induced cells were seeded into PVA-n-HA/PA6 separately, and the constructs were implanted into the rabbit muscle pouch for upto 12 weeks. Ectopic neocartilage formation in the PVA layer and reconstitution of the subchondral bone which remained confined within the n-HA/PA6 layer with the alteration of the cellular phenotype were identified with Masson's trichrome stain. Simultaneously, the RT-PCR results confirmed the expression of specific extracellular matrix (ECM) markers for cartilaginous tissue, such as collagen type II (Col-II), or alternatively, markers for osteoid tissue, such as collagen type I (Col-I) at the corresponding layers. During ectopic implantation, the underlying subchondral bone layer was completely integrated with the cartilage layer. The result from the ectopic osteochondral scaffolds implantation suggests that PVA-n-HA/PA6 with induced BMSCs is a possible substitute with potential in cartilage repair strategies.     

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.     

An in vitro study of collagen hydrogel to induce the chondrogenic differentiation of mesenchymal stem cells

It is controversial whether a biomaterial itself, rather than addition of any exogenous growth factor, could induce mesenchymal stem cells (MSCs) to differentiate into chondrogenic lineage, further to regenerate cartilage. Previous studies have shown that collagen-based hydrogel could induce MSCs to differentiate into chondrocytes in vivo but the in vitro studies only have a few reports. The evidence that biomaterials could induce chondrogenesis is not adequate. In this study, we tried to address whether type I collagen hydrogel has chondro-inductive capability in vitro and how this scaffold induces MSCs to generate cartilage tissue without exogenous growth factors in the culture medium. We encapsulated neonatal rabbit bone marrow mesenchymal stem cells (BMSCs) in type I collagen hydrogel homogeneously or implanted cell aggregates in hydrogel, and cultured them in nonchondrogenic inductive media. After at least 28 days culture, cells in the homogeneous group were tending to chondrogenic differentiation while cell density was high, and cells in the aggregate group have almost gone through chondrogenesis and formed neo-cartilage tissue with abundant specific extracellular matrix (ECM) deposition. These results indicate collagen hydrogel has inherent inductivity for the chondrogenic differentiation of BMSCs, and the optimum specification and tissue formation were accompanied with local high cell density. This research suggests a feasible strategy to induce the chondro differentiation of BMSCs independent of exogenous growth factors, which may greatly contribute to clinical cartilage regeneration. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A 100A: 2717-2725, 2012.     

In vivo bone generation via the endochondral pathway on three-dimensional electrospun fibers

A new concept of generating bone tissue via the endochondral route might be superior to the standard intramembranous ossification approach. To implement the endochondral approach, suitable scaffolds are required to provide a three-dimensional (3-D) substrate for cell population and differentiation, and eventually for the generation of osteochondral tissue. Therefore, a novel wet-electrospinning system, using ethanol as the collecting medium, was exploited in this study to fabricate a cotton-like poly(lactic-co-glycolic acid)/poly(ε-caprolactone) scaffold that consisted of a very loose and uncompressed accumulation of fibers. Rat bone marrow cells were seeded on these scaffolds and chondrogenically differentiated in vitro for 4 weeks followed by subcutaneous implantation in vivo for 8 weeks. Cell pellets were used as a control. A glycosaminoglycan assay and Safranin O staining showed that the cells infiltrated throughout the scaffolds and deposited an abundant cartilage matrix after in vitro chondrogenic priming. Histological analysis of the in vivo samples revealed extensive new bone formation through the remodeling of the cartilage template. In conclusion, using the wet-electrospinning method, we are able to create a 3-D scaffold in which bone tissue can be formed via the endochondral pathway. This system can be easily processed for various assays and histological analysis. Consequently, it is more efficient than the traditional cell pellets as a tool to study endochondral bone formation for tissue engineering purposes.     

The internalized CdSe/ZnS quantum dots impair the chondrogenesis of bone marrow mesenchymal stem cells

Mesenchymal stem cells (MSCs) are capable of differentiating into multiple cell lineages and are useful for therapeutic applications. Labeling the MSCs with fluorescent probes is beneficial in tracing the fate of MSCs after implantation. We have introduced the CdSe/ZnS quantum dots (QDs) into the human bone marrow MSCs and examined the effects of QDs on the proliferation and chondrogenesis of the cells. The internalized QDs were found localized in perinuclear regions and remained there after a number of cell passages. The presence of QDs did not affect the proliferation of cells or the size of chondrospheres formed, when subjected to chondrogenesis induction. However, the expression of mRNA and protein of type II collagen and aggrecan in the chondrospheres was significantly inhibited in cells labeled with QDs, suggesting impaired chondrogenesis. Our results that the presence of QDs interferes with the chondrogenic differentiation of MSCs raise concerns in using the QDs as fluorescence tracers for stem cells.     

The role of trabecular demineralized bone in combination with perichondrium in the generation of cartilage grafts

The use of a composite graft of bovine trabecular demineralized bone matrix (DBM) and perichondrium has been found a reliable method for in vivo generation of cartilage. In the present study, the mechanism whereby this commercially available matrix increases cartilage formation was investigated. First, the time course of cartilage formation in vivo, in the combined implant of perichondrium and DBM in the rabbit ear was studied, with special focus on tissue reactions to DBM. DBM was colonized by macrophages from day 3 post-operatively, reaching a maximum after 2 weeks. Only a minimal number of neutrophils was found. After 3 weeks the DBM appeared to be resorbed. In the first week the DBM was invaded with chondroblasts, and chondrogenesis occurred between the first and second week of implantation. After 3 weeks, the initially formed islets of cartilage had fused. Next, the chondrogenic capacity of DBM itself was investigated by implantation of DBM without perichondrium. This never resulted in cartilage formation. Immunohistochemistry showed only a faint staining of the DBM for growth factors. This indicates a minimal chondrogenic effect of DBM alone and the requirement of perichondrium as cell provider. In order to define the conditions which cause chondrogenesis in composites of perichondrium and DBM, a series of in vitro culture experiments was performed in which the in vivo situation was mimicked step by step. The basic condition was perichondrium cultured in medium with 10% FCS. In this condition, cartilage formation was variable. Because in the in vivo situation both DBM and macrophages can release growth factors, the effect of IGF1, TGFbeta2 or OP1 added to the culture medium was tested. Neither the incidence nor the amount of cartilage formation was stimulated by addition of growth factors. Perichondrium wrapped around DBM in vitro gave cartilage formation in the perichondrium but the incidence and amount were not significantly stimulated compared to cultures of perichondrium without DBM. However, cartilage-like cells were found in the DBM suggesting an effect of DBM on perichondrium-derived cells. Finally, macrophages and/or blood were added to the composite DBM-perichondrium to mimic the in vivo situation as close as possible. However, no effect of this treatment was found. In conclusion, this study indicates that DBM itself has few chondrogenic qualities but functions merely as a spacer for cell ingrowth. The fast resorption of DBM by macrophages in vivo seems of importance for the cartilage forming process, but in vitro the presence of macrophages (in combination with blood) could not enhance chondrogenesis.