Higher Chondrogenic Potential of Extracellular Vesicles Derived from Mesenchymal Stem Cells Compared to Chondrocytes-EVs In Vitro

The inability of cartilage to self-repair necessitates an effective therapeutic approach to restore damaged tissues. Extracellular vesicles (EVs) are attractive options because of their roles in cellular communication and tissue repair where they regulate the cellular processes of proliferation, differentiation, and recruitment. However, it is a challenge to determine the relevant cell sources for isolation of EVs with high chondrogenic potential. The current study aims to evaluate the chondrogenic potential of EVs derived from chondrocytes (Cho-EV) and mesenchymal stem cells (MSC-EV). The EVs were separately isolated from conditioned media of both rabbit bone marrow MSCs and chondrocyte cultures. The isolated vesicles were assessed in terms of size, morphology, and surface marker expression. The chondrogenic potential of MSCs in the presence of different concentrations of EVs (50, 100, and 150 μg/ml) was evaluated during 21 days, and chondrogenic surface marker expressions were checked by qRT-PCR and histologic assays. The extracted vesicles had a spherical morphology and a size of 44.25 ± 8.89 nm for Cho-EVs and 112.1 ± 10.10 nm for MSC-EVs. Both groups expressed the EV-specific surface markers CD9 and CD81. Higher expression of chondrogenic specified markers, especially collagen type II (COL II), and secretion of glycosaminoglycans (GAGs) and proteoglycans were observed in MSCs treated with 50 and 100 μg/ml MSC-EVs compared to the Cho-EVs. The results from the use of EVs, particularly MSC-EVs, with high chondrogenic ability will provide a basis for developing therapeutic agents for cartilage repair.

Inhibition of PPARγ Gene Particle Downregulates the Differentiation of Rabbit Bone Marrow Mesenchymal Stem Cells into Adipocytes

This study aims to evaluate the effect of peroxisome proliferator-activated receptor (PPAR) γ gene inhibition on the adipogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs). Primary BMSCs were isolated from rabbit bone marrow, cultured, and the markers of BMSCs on cell’s surface were analyzed using flow cytometry. The experiment involved five groups, namely, control: untreated BMSCs; model: BMSCs treated with ethanol; empty siRNA: BMSCs treated with ethanol + empty siRNA; PPARγ: BMSCs treated with ethanol + PPARγ siRNA; and PPARγ inhibitor: BMSCs treated with ethanol + T0070907. RT-PCR and Western blotting were used to detect changes in the expression level of PPARγ, PETALA2 (AP2), lipoprotein lipase (LPL), fatty acid transport protein (FATP) 1, and fatty acid transporter (FAT). Adipocyte count and triacylglycerol content of the model and the empty siRNA groups were considerably greater than the control group ( P < 0.01 ). After the inhibition with PPARγ or T0070907, adipocyte count and triacylglycerol content of the PPARγ and T0070907 groups were significantly reduced ( P < 0.01 ), with no statistically significantly difference than the control group ( P > 0.05 ). The expression levels of PPARγ gene and protein in the model and empty siRNA groups were ominously enhanced than the control group ( P < 0.01 ), and after inhibition with PPARγ or T0070907, the PPARγ gene or protein expression level of PPARγ and T0070907 groups significantly reduced ( P < 0.01 ), with no statistically significance difference compared to the control group ( P > 0.05 ). The expression levels of Ap2, LPL, FATP1, and FAT genes in the model and empty siRNA groups were considerably greater compared to the control group ( P < 0.01 ). Inhibition with PPARγ or T0070907 in the PPARγ and T0070907 groups, respectively, lead to significantly reduced expression levels of adipogenic genes ( P < 0.01 ), with no statistically significance difference compared to the control ( P > 0.05 ). Inhibition of PPARγ gene downregulates the differentiation of BMSCs into adipocytes, indicating its putative role in the expression of adipogenic genes.

Regeneration of Bone Defects in a Rabbit Femoral Osteonecrosis Model Using 3D-Printed Poly (Epsilon-Caprolactone)/Nanoparticulate Willemite Composite Scaffolds

Steroid-associated osteonecrosis (SAON) is a chronic disease that leads to the destruction and collapse of bone near the joint that is subjected to weight bearing, ultimately resulting in a loss of hip and knee function. Zn2+ ions, as an essential trace element, have functional roles in improving the immunophysiological cellular environment, accelerating bone regeneration, and inhibiting biofilm formation. In this study, we reconstruct SAON lesions with a three-dimensional (3D)-a printed composite made of poly (epsilon-caprolactone) (PCL) and nanoparticulate Willemite (npW). Rabbit bone marrow stem cells were used to evaluate the cytocompatibility and osteogenic differentiation capability of the PCL/npW composite scaffolds. The 2-month bone regeneration was assessed by a Micro-computed tomography (micro-CT) scan and the expression of bone regeneration proteins by Western blot. Compared with the neat PCL group, PCL/npW scaffolds exhibited significantly increased cytocompatibility and osteogenic activity. This finding reveals a new concept for the design of a 3D-printed PCL/npW composite-based bone substitute for the early treatment of osteonecrosis defects.

Mesenchymal Stromal Cells Enhance Vascularization and Epithelialization within 7 Days after Gingival Augmentation with Collagen Matrices in Rabbits

Soft gingival tissue deficiency remains a severe problem leading to postoperative recession, peri-implantitis, and bone resorption. The use of collagen matrices does not always lead to complete rebuilding of the gingiva volume. The application of mesenchymal stromal cells (MSCs) simultaneously with collagen materials represents a promising approach for the restoration of soft gingival tissues. However, short-term effects of MSCs-enriched collagen grafts after gingival augmentation have not yet been studied properly. Mucograft and Mucoderm matrices were implanted in rabbits (n = 12) simultaneously with the intraoperative injection of rabbit bone marrow-derived mesenchymal stromal cells (BM-MSCs) or without cells. Collagen matrices were implanted under the flap or by the surface technique without intentional primary closure. The samples were harvested seven days after implantation, histological staining with hematoxylin and eosin, and immunohistochemical staining for VEGF, IGF1, and TGF were performed. The use of Mucoderm led to better augmentation outcomes on day 7 compared with Mucograft (p < 0.0001). Gingival augmentation in combination with the local administration of BM-MSCs led to better regeneration of the soft gingival tissues independently of the type of implanted collagen matrices (p < 0.0001). Furthermore, injection of BM-MSCs significantly enhanced gingival vascularization and epithelization with a clear positive correlation between vascular growth and epithelial response. Administration of BM-MSCs in combination with various collagen materials may potentially improve gingiva regeneration.

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.

Clinical Efficacy of Polycaprolactone β-Calcium Triphosphate Composite for Osteoconduction in Rabbit Bone Defect Model

The combination of β-tricalcium phosphate (β-TCP) with polycaprolactone (PCL) has been considered a promising strategy for designing scaffolds for bone grafting. This study incorporated PCL with commercially available β-TCP (OsteoceraTM) to fabricate an injectable bone substitute and evaluate the effect of PCL on compressive strength and setting time of the hydraulic cement. The mechanical testing was compliant with the ASTM D695 and ASTM C191-13 standards. Results showed that PCL-TCP composite presented a well-defined architecture with uniform pore distribution and a significant increase in compressive strength compared with β-TCP alone. Eighteen rabbits, each with two surgically created bone defects, were treated using the PCL-TCP composites. The composite materials were resorbed and replaced by newly formed bone tissue. Both PCL-TCP and β-TCP demonstrated equivalent clinical effects on osteoconduction property in terms of the percentage of newly formed bone area measured by histomorphometric analysis. PCL-TCP was proven to be as effective as the commercially available β-TCP scaffold (OsteoceraTM).

The Effect of Stromal-Derived Factor 1α on Osteoinduction Properties of Porous β-Tricalcium Phosphate Bioceramics

β-Tricalcium phosphate (TCP) is a type of bioceramic material which is commonly used for hard tissue repair and famous of its remarkable biocompatibility and osteoconductivity with similar composition to natural bone. However, TCP lacks osteoindcutive properties. Stromal-derived factor 1α (SDF-1α) can promote bone regeneration with excellent osteoinduction effect. In this study, SDF-1α was loaded into TCP to investigate the in vitro effects of SDF-1α on the osteoinductive properties of TCP. In vitro studies showed that SDF-1α/TCP scaffold significantly stimulated the expression of osteopontin and osteocalcin. As to the in vivo studies, the rabbit bone defect model showed that SDF-1α stimulated more new bone formation. In conclusion, SDF-1α/TCP bioceramic scaffolds could further promote bone regeneration compared to pure TCP bioceramics.