Cartilage Regeneration Characteristics of Human and Goat Auricular Chondrocytes

Although cartilage regeneration technology has achieved clinical breakthroughs, whether auricular chondrocytes (AUCs) represent optimal seed cells to achieve stable cartilage regeneration is not clear. In this study, we systematically explore biological behaviors of human- and goat-derived AUCs during in vitro expansion as well as cartilage regeneration in vitro and in vivo. To eliminate material interference, a cell sheet model was used to evaluate the feasibility of dedifferentiated AUCs to re-differentiate and regenerate cartilage in vitro and in vivo. We found that the dedifferentiated AUCs could re-differentiate and regenerate cartilage sheets under the chondrogenic medium system, and the generated chondrocyte sheets gradually matured with increased in vitro culture time (2, 4, and 8 weeks). After the implantation of cartilage sheets with different in vitro culture times in nude mice, optimal neocartilage was formed in the group with 2 weeks in vitro cultivation. After in vivo implantation, ossification only occurred in the group with goat-regenerated cartilage sheet of 8 weeks in vitro cultivation. These results, which were confirmed in human and goat AUCs, suggest that AUCs are ideal seed cells for the clinical translation of cartilage regeneration under the appropriate culture system and culture condition.

Effect of Activated Platelet-Rich Plasma on Chondrogenic Differentiation of Rabbit Bone Marrow-Derived Mesenchymal Stem Cells

We aimed to evaluate the effect of activated platelet-rich plasma (PRP) on proliferation and chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Six mature male rabbits were included in this study. PRP was obtained by two-step centrifugation from whole blood, and it was activated using CaCl2 solution. BMSCs were isolated and proliferated from bone marrow of rabbits and characterized by flow cytometry. Passage 3 BMSCs were cultured in high-glucose Dulbecco’s modified Eagle’s medium (HG-DMEM) with the four different compositions for consecutive 7 days, including 10% fetal bovine serum, 5% PRP, 10% PRP, and 15% PRP. Cell counting assays were performed to evaluate the cell proliferation of BMSCs. BMSCs ( 5 × 10 5 cells/well in 6-well plates) were induced in four conditions for 21 days to chondrogenic differentiation evaluation, including commercial chondrogenic medium (control), 5% PRP (HG-DMEM+5% PRP), 10% PRP (HG-DMEM+10% PRP), and 15% PRP (HG-DMEM+15% PRP). The gene expression levels of ACAN, COL2A1, and SOX9 in pellets were detected. Morphological and pathological assessments were performed by the blind observer. After purifying, the percentages of cells with CD105(+)/CD34(−) and CD44(+)/CD45(−) were 96.5% and 92.9%, respectively. The proliferation of BMSCs was enhanced in all groups, and 10% PRP revealed more significant outcome than the others from day 5. The levels of ACAN, COL2A1, and SOX9 were lower in the three PRP groups than control group, but the levels of ACAN and SOX9 were higher in 10% PRP group than 5% and 15% PRP groups. Histological examinations showed that 10% PRP-treated pellets had more regular appearance, larger size, and abundant extracellular matrix than 5% or 10% PRP groups, but still inferior to commercial chondrogenic medium. In conclusion, our results show that PRP may enhance the proliferation of rabbit BMSCs. However, PRP have limited effect on chondrogenic differentiation in comparison with commercial chondrogenic medium in pellets culture.

Inability of Low Oxygen Tension to Induce Chondrogenesis in Human Infrapatellar Fat Pad Mesenchymal Stem Cells

ObjectiveArticular cartilage of the knee joint is avascular, exists under a low oxygen tension microenvironment, and does not self-heal when injured. Human infrapatellar fat pad-sourced mesenchymal stem cells (IFP-MSC) are an arthroscopically accessible source of mesenchymal stem cells (MSC) for the repair of articular cartilage defects. Human IFP-MSC exists physiologically under a low oxygen tension (i.e., 1–5%) microenvironment. Human bone marrow mesenchymal stem cells (BM-MSC) exist physiologically within a similar range of oxygen tension. A low oxygen tension of 2% spontaneously induced chondrogenesis in micromass pellets of human BM-MSC. However, this is yet to be demonstrated in human IFP-MSC or other adipose tissue-sourced MSC. In this study, we explored the potential of low oxygen tension at 2% to drive the in vitro chondrogenesis of IFP-MSC. We hypothesized that 2% O2 will induce stable chondrogenesis in human IFP-MSC without the risk of undergoing endochondral ossification at ectopic sites of implantation.MethodsMicromass pellets of human IFP-MSC were cultured under 2% O2 or 21% O2 (normal atmosphere O2) in the presence or absence of chondrogenic medium with transforming growth factor-β3 (TGFβ3) for 3 weeks. Following in vitro chondrogenesis, the resulting pellets were implanted in immunodeficient athymic nude mice for 3 weeks.ResultsA low oxygen tension of 2% was unable to induce chondrogenesis in human IFP-MSC. In contrast, chondrogenic medium with TGFβ3 induced in vitro chondrogenesis. All pellets were devoid of any evidence of undergoing endochondral ossification after subcutaneous implantation in athymic mice.

Type II Collagen Sponges Facilitate Tendon Stem/Progenitor Cells to Adopt More Chondrogenic Phenotypes and Promote the Regeneration of Fibrocartilage-Like Tissues in a Rabbit Partial Patellectomy Model

ObjectiveFibrocartilage transition zone (FC) is difficult to regenerate after surgical re-attachment of tendon to bone. Here, we investigated whether type II collagen-sponges (CII-sponges) facilitated tendon stem/progenitor cells (TSPCs) to adopt chondrogenic phenotypes and further observed if this material could increase the FC areas in bone-tendon junction (BTJ) injury model.MethodsCII-sponges were made as we previously described. The appearance and pore structure of CII-sponges were photographed by camera and microscopies. The viability, proliferation, and differentiation of TSPCs were examined by LIVE/DEAD assay, alamarBlue, and PKH67 in vitro tracking. Subsequently, TSPCs were seeded in CII-sponges, Matrigel or monolayer, and induced under chondrogenic medium for 7 or 14 days before being harvested for qPCR or being transplanted into nude mice to examine the chondrogenesis of TSPCs. Lastly, partial patellectomy (PP) was applied to establish the BTJ injury model. CII-sponges were interposed between the patellar fragment and tendon, and histological examination was used to assess the FC regeneration at BTJ after surgery at 8 weeks.ResultsCII-sponges were like sponges with interconnected pores. TSPCs could adhere, proliferate, and differentiate in this CII-sponge up to 14 days at least. Both qPCR and immunostaining data showed that compared with TSPCs cultured in monolayer or Matrigel, cells in CII-sponges group adopted more chondrogenic phenotypes with an overall increase of chondrocyte-related genes and proteins. Furthermore, in PP injured model, much more new formed cartilage-like tissues could be observed in CII-sponges group, evidenced by a large amount of positive proteoglycan expression and typical oval or round chondrocytes in this area.ConclusionOur study showed that CII-sponges facilitated the TSPCs to differentiate toward chondrocytes and increased the area of FCs, which suggests that CII-sponges are meaningful for the reconstruction of FC at bone tendon junction. However, the link between the two phenomena requires further research and validation.

Effects of Normal Synovial Fluid and Interferon Gamma on Chondrogenic Capability and Immunomodulatory Potential Respectively on Equine Mesenchymal Stem Cells

Synovial fluid contains cytokines, growth factors and resident mesenchymal stem cells (MSCs). The present study aimed to (1) determine the effects of autologous and allogeneic synovial fluid on viability, proliferation and chondrogenesis of equine bone marrow MSCs (BMMSCs) and (2) compare the immunomodulatory properties of equine synovial fluid MSCs (SFMSCs) and BMMSCs after stimulation with interferon gamma (INF-γ). To meet the first aim of the study, the proliferation and viability of MSCs were evaluated by MTS and calcein AM staining assays. To induce chondrogenesis, MSCs were cultured in a medium containing TGF-β1 or different concentrations of synovial fluid. To meet the second aim, SFMSCs and BMMSCs were stimulated with IFN-γ. The concentration of indoleamine-2,3-dioxygenase (IDO) and nitric oxide (NO) were examined. Our results show that MSCs cultured in autologous or allogeneic synovial fluid could maintain proliferation and viability activities. Synovial fluid affected chondrocyte differentiation significantly, as indicated by increased glycosaminoglycan contents, compared to the chondrogenic medium containing 5 ng/mL TGF-β1. After culturing with IFN-γ, the conditioned media of both BMMSCs and SFMSCs showed increased concentrations of IDO, but not NO. Stimulating MSCs with synovial fluid or IFN-γ could enhance chondrogenesis and anti-inflammatory activity, respectively, suggesting that the joint environment is suitable for chondrogenesis.

The effect of doxycycline and collagenase A on the differentiation of mesenchymal stem cells towards a nucleus pulposus phenotype

Intervertebral disc (IVD) degeneration costs the healthcare system billions of dollars annually and leads to reduced quality of life. Current treatments are invasive and primarily focus on symptom relief rather than repair. This study aimed to facilitate the development of an injectable therapy using chondrogenically differentiated mesenchymal stem cells (MSCs) in the absence of collagen II deposition. Briefly, pelleted MSCs were cultivated in chondrogenic medium and were supplemented with collagenase A or doxycycline in order to inhibit collagen assembly. Results indicated that collagenase A and doxycycline treatment had no negative effects on DNA or proteoglycan content. Collagenase A at all concentrations affected collagen content, as did doxycycline at low concentrations. Furthermore, preliminary gene expression studies for nucleus pulposus markers showed that collagenase A and doxycycline may have some effect on terminal differentiation of MSCs in chondrogenic medium. Overall, the findings suggest that collagenase A and doxycycline supplementation can be used to inhibit collagen formation, thereby facilitating the further development of an injectable therapy for IVD repair.

The effect of doxycycline and collagenase A on the differentiation of mesenchymal stem cells towards a nucleus pulposus phenotype

Intervertebral disc (IVD) degeneration costs the healthcare system billions of dollars annually and leads to reduced quality of life. Current treatments are invasive and primarily focus on symptom relief rather than repair. This study aimed to facilitate the development of an injectable therapy using chondrogenically differentiated mesenchymal stem cells (MSCs) in the absence of collagen II deposition. Briefly, pelleted MSCs were cultivated in chondrogenic medium and were supplemented with collagenase A or doxycycline in order to inhibit collagen assembly. Results indicated that collagenase A and doxycycline treatment had no negative effects on DNA or proteoglycan content. Collagenase A at all concentrations affected collagen content, as did doxycycline at low concentrations. Furthermore, preliminary gene expression studies for nucleus pulposus markers showed that collagenase A and doxycycline may have some effect on terminal differentiation of MSCs in chondrogenic medium. Overall, the findings suggest that collagenase A and doxycycline supplementation can be used to inhibit collagen formation, thereby facilitating the further development of an injectable therapy for IVD repair.

Decellularized Articular Cartilage Microparticles for Expansion of Mesenchymal Stem Cells and Zonal Regeneration of Articular Cartilage

Introduction: The objective was to create multilayer cellular constructs using fetal or adult, decellularized articular cartilage in particulate form as microcarriers for expansion and fusion of mesenchymal stem cells (MSCs) to regenerate the stratified structure of articular cartilage. Methods: Porous microparticles (CMPs) generated from decellularized fetal or adult bovine articular cartilage were used as microcarriers for expansion of human MSCs. The CMP expanded MSCs (CMP-MSCs) were used to generate injectable hydrogels or preformed multilayer constructs for articular cartilage regeneration. In the injectable approach, CMP-MSCs were suspended in alginate gel, crosslinked with calcium chloride, and incubated in chondrogenic medium to generate an injectable regenerative construct. In the preformed approach, fetal or adult CMP-MSCs were suspended in a culture medium, allowed to settle sequentially by the force of gravity, and fused by incubation in chondrogenic medium to generate multilayer cell sheets. The constructs were characterized with respect to compressive modulus, cellularity, and expression of chondrogenic markers. Results: Human MSCs expanded on fetal or adult CMPs in basal medium maintained the expression of mesenchymal markers. The injectable CMP-MSCs hydrogels had significantly higher expression of chondrogenic markers and compressive modulus after four weeks incubation in chondrogenic medium compared to MSCs directly encapsulated in alginate gel; preformed CMP-MSCs cell sheets had significantly higher compressive modulus and expression of chondrogenic markers compared to MSCs in the pellet culture. Conclusion: The preformed cell sheet approach is potentially useful for creating multilayer constructs by sequential gravitational settling of CMP-MSCs to mimic the stratified structure of articular cartilage.

Effect of Activated Platelet-Rich Plasma on Chondrogenic Differentiation of Rabbit Bone Marrow-Derived Mesenchymal Stem Cells

Background Platelet-rich plasma (PRP) has revealed benefits in tissue repair and regeneration, however, the effect of PRP on proliferation and chondrogenesis of mesenchymal stem cells remains controversial.This study is to evaluate the effect of different concentration PRP on proliferation and chondrogenic differentiation of rabbit bone marrow-derived mesenchymal stem cells(BMSCs). Methods PRP was obtained by centrifugation and activation, in which growth factors and cytokines were detected. BMSCs were isolated from rabbit bone marrow and characterized by flow cytometry. About 5 × 103 BMSCs were cultured in high glucose Dulbecco’s modified Eagle’s medium (HG-DMEM) with 4 different compositions: 10% fetal bovine serum, 5%PRP, 10%PRP, and 15%PRP for consecutive 7 days. Cell counting assays were performed on days 1, 3, 5, and 7 to evaluate the BMSCs proliferation. In chondrogenic differentiation, high-density cell pellets composed of 5 × 105 BMSCs were induced in 4 conditions: commercial chondrogenic medium (control), 5%PRP (HG-DMEM + 5%PRP), 10%PRP (HG-DMEM + 10%PRP), and 15%PRP (HG-DMEM + 15%PRP) for 21 days. The gene expression levels of aggrecan (ACAN), collagen type Ⅱ Alpha 1 (COL2A1), and SRY-Box Transcription Factor 9 (SOX9) in pellets were detected. Histological assessments were performed by morphologically observation and pathological stain. Independent-samples t-test and one-way analysis of variance were used in statistical analyses. Results The concentrations of growth factors and cytokines were elevated in PRP. BMSCs proliferation was enhanced in all groups, and 10% PRP revealed more obvious outcome than the others from day 5. In chondrogenic differentiation, the levels of ACAN, COL2A1, and SOX9 were lower in 3 PRP groups than control, but ACAN and SOX9 was higher in 10% PRP group than 5% and 15%. Histological examinations showed 10% PRP-treated pellets showed more regular appearance, larger size, and abundant extracellular matrix than 5% and 10% groups, but still inferior to commercial chondrogenic medium. Conclusions PRP may enhance the proliferation of rabbit BMSCs, while with limited effect on chondrogenic differentiation compared with commercial chondrogenic medium in pellets culture. Whether optimizing and modifying PRP components would lead to satisfying chondrogenesis of BMSCs, it is deserved furthermore study.

Which Culture System Is better for Chondrogenesis of Adipose-Derived Stem Cells ; Pellet or Micromass?

Background and Aims: The current study was conducted to compare the expression levels of collagen type Π and X during chondrogenesis of human adipose-derived mesenchymal stem cells (hADMSCs) pellet and micromass cultures. Materials and Methods: Extracted hADMSCs were cultured until three passages and then transferred to pellet and micromass cultures in the experimental groups of day 7 and day14. For pellet and micromass cultures, aliquots of 5×105 cells/ml were centrifuged and respectively cultured in the conical tubes and droplets (12.5 µl) of the 24-well plates containing chondrogenic medium. Realtime-polymerase chain reaction technique was performed for gene expression levels. Results: Increased expression of collagen type Π was shown in micromass day14 compared to micromass day 7, pellet day 14 (p<0.01) and pellet day 7 (p<0.001). Also, an increased expression of collagen type Π was seen in micromass day 7 and pellet day 14 compared to pellet day 7 (p< 0.05). Expression of collagen type X increased in pellet day 14 compared to micromass on days 7 and 14 (p<0.001, p<0.01) and pellet day14 compared to pellet day7 (p< 0.05). An increased expression of collagen type X was shown in pellet day 7 compared to micromass on days 7 and 14 (p<0.05). Conclusions: According to the results, higher expression of collagen type Π and lower expression of collagen type X in micromass cultures that are prepared by cell suspension play a better role during cellular condensation that leads to the formation of large nodules exhibiting cartilage-like morphology, suggests a higher efficiency for micromass cultures.