The recruitment of bone marrow CD34- mesenchymal stem- and progenitor cells (MSC) and their subsequent differentiation into distinct tissues is the precondition for in situ tissue engineering. The objective of this study was to determine the entire chemokine receptor expression profile of human MSC and to investigate their chemotactic response to the selected chemokines CCL2, CXCL8 and CXCL12. Human MSC were isolated from iliac crest bone marrow aspirates and showed a homogeneous population presenting a typical MSC-related cell surface antigen profile (CD14-, CD34-, CD44+, CD45-, CD166+, SH-2+). The expression profile of all 18 chemokine receptors was determined by real-time PCR and immunohistochemistry. Both methods consistently demonstrated that MSC express CC, CXC, C and CX(3)C receptors. Gene expression and immunohistochemical analysis documented that MSC express chemokine receptors CCR2, CCR8, CXCR1, CXCR2 and CXCR3. A dose-dependent chemotactic activity of CXCR4 and CXCR1/CXCR2 ligands CXCL12 and CXCL8 (interleukin-8) was demonstrated using a 96-well chemotaxis assay. In contrast, the CCR2 ligand CCL2 (monocyte chemoattractant protein-1, MCP-1) did not recruited human MSC. In conclusion, we report that the chemokine receptor expression profile of human MSC is much broader than known before. Furthermore, for the first time, we demonstrate that human MSC migrate upon stimulation with CXCL8 but not CCL2. In combination with already known data on MSC recruitment and differentiation these are promising results towards in situ regenerative medicine approaches based on guiding of MSC to sites of degenerated tissues.

In cartilage repair, platelet-rich plasma (PRP) is used in one-step approaches utilizing microfracture and matrix-induced chondrogenesis procedures, bone marrow-derived cell transplantation, or intra-articular injection. The aim of our study was to evaluate the effect of human PRP on the migration and chondrogenic differentiation of human subchondral progenitors. Human progenitors were derived from subchondral cortico-spongious bone (CSP), were analyzed for their migration capacity upon PRP treatment in 96-well chemotaxis assays and cultured in high-density pellet cultures under serum-free conditions in the presence of 5% PRP. Chemotaxis assays showed that 0.1-100% PRP significantly (p < 0.05) stimulate the migration of CSP compared to untreated controls. Histological staining of proteoglycan and immuno-staining of type II collagen indicated that progenitors stimulated with PRP show significantly increased cartilage matrix formation compared to untreated progenitors. Real-time gene expression analysis of typical chondrocyte marker genes as well as osteogenic and adipogenic markers like osteocalcin and fatty acid binding protein showed that PRP induces the chondrogenic differentiation sequence of human progenitors in high-density pellet cultures, while osteogenic or adipogenic differentiation was not evident. These results suggest that human PRP may enhance the migration and stimulate the chondrogenic differentiation of human subchondral progenitor cells known from microfracture. ß

The aim of our study was to evaluate the mid-term outcome of a cell-free polymer-based cartilage repair approach in a sheep cartilage defect model in comparison to microfracture treatment. Cell-free, freeze-dried implants (chondrotissue 1 ) made of a poly-glycolic acid (PGA) scaffold and hyaluronan were immersed in autologous serum and used for covering microfractured full-thickness articular cartilage defects of the sheep (n ¼ 4). Defects treated with microfracture only served as controls (n ¼ 4). Six months after implantation, cartilage implants and controls were analyzed by immunohistochemical staining of type II collagen, histological staining of proteoglycans, and histological scoring. Histological analysis showed the formation of a cartilaginous repair tissue rich in proteoglycans. Histological scoring documented significant improvement of repair tissue formation when the defects were covered with the cell-free implant, compared to controls treated with microfracture. Immunohistochemistry showed that the cell-free implant induced cartilaginous repair tissue and type II collagen. Controls treated with microfracture showed marginal formation of a mixed-type repair tissue consisting of cartilaginous tissue and fibro-cartilage. Covering of microfractured defects with the cell-free polymer-based cartilage implant is suggested to be a promising treatment option for cartilage defects and improves the regeneration of articular cartilage. ß

IntroductionMesenchymal stem cells (MSC) are highly attractive for use in cartilage regeneration. To date, MSC are usually recruited from subchondral bone marrow using microfracture. Recent data suggest that isolated cells from adult human articular cartilage, which express the combination of the cell-surface markers CD105 and CD166, are multi-potent mesenchymal progenitor cells (MPC) with characteristics similar to MSC. MPC within the cartilage matrix, the target of tissue regeneration, may provide the basis for in situ regeneration of focal cartilage defects. However, there is only limited information concerning the presence/abundance of CD105+/CD166+ MPC in human articular cartilage. The present study therefore assessed the relative percentage and particularly the zonal distribution of cartilage MPC using the markers CD105/CD166.MethodsSpecimens of human osteoarthritic (OA; n = 11) and normal (n = 3) cartilage were used for either cell isolation or immunohistochemistry. Due to low numbers, isolated cells were expanded for 2 weeks and then analyzed by flow cytometry (FACS) or immunofluorescence in chamber slides for the expression of CD105 and CD166. Following immunomagnetic separation of CD166+/- OA cells, multi-lineage differentiation assays were performed. Also, the zonal distribution of CD166+ cells within the matrix of OA and normal cartilage was analyzed by immunohistochemistry.ResultsFACS analysis showed that 16.7 ± 2.1% (mean ± SEM) of OA and 15.3 ± 2.3 of normal chondrocytes (n.s.) were CD105+/CD166+ and thus carried the established MPC marker combination. Similarly, 13.2% ± 0.9% and 11.7 ± 2.1 of CD105+/CD166+cells, respectively, were identified by immunofluorescence in adherent OA and normal chondrocytes. The CD166+ enriched OA cells showed a stronger induction of the chondrogenic phenotype in differentiation assays than the CD166+ depleted cell population, underlining the chondrogenic potential of the MPC. Strikingly, CD166+ cells in OA and normal articular cartilage sections (22.1 ± 1.7% and 23.6% ± 1.4%, respectively; n.s.) were almost exclusively located in the superficial and middle zone.ConclusionsThe present results underline the suitability of CD166 as a biomarker to identify and, in particular, localize and/or enrich resident MPC with a high chondrogenic potential in human articular cartilage. The percentage of MPC in both OA and normal cartilage is substantially higher than previously reported, suggesting a yet unexplored reserve capacity for regeneration.

The aim of this project was to investigate the in vitro osteogenic potential of human mesenchymal progenitor cells in novel matrix architectures built by means of a three-dimensional bioresorbable synthetic framework in combination with a hydrogel. Human mesenchymal progenitor cells (hMPCs) were isolated from a human bone marrow aspirate by gradient centrifugation. Before in vitro engineering of scaffold-hMPC constructs, the adipogenic and osteogenic differentiation potential was demonstrated by staining of neutral lipids and induction of bone-specific proteins, respectively. After expansion in monolayer cultures, the cells were enzymatically detached and then seeded in combination with a hydrogel into polycaprolactone (PCL) and polycaprolactone-hydroxyapatite (PCL-HA) frameworks. This scaffold design concept is characterized by novel matrix architecture, good mechanical properties, and slow degradation kinetics of the framework and a biomimetic milieu for cell delivery and proliferation. To induce osteogenic differentiation, the specimens were cultured in an osteogenic cell culture medium and were maintained in vitro for 6 weeks. Cellular distribution and viability within three-dimensional hMPC bone grafts were documented by scanning electron microscopy, cell metabolism assays, and confocal laser microscopy. Secretion of the osteogenic marker molecules type I procollagen and osteocalcin was analyzed by semiquantitative immunocytochemistry assays. Alkaline phosphatase activity was visualized by p-nitrophenyl phosphate substrate reaction. During osteogenic stimulation, hMPCs proliferated toward and onto the PCL and PCL-HA scaffold surfaces and metabolic activity increased, reaching a plateau by day 15. The temporal pattern of bone-related marker molecules produced by in vitro tissue-engineered scaffold-cell constructs revealed that hMPCs differentiated better within the biomimetic matrix architecture along the osteogenic lineage.

To create artificial cartilage in vitro, mimicking the function of native extracellular matrix (ECM) and morphological cartilage‐like shape is essential. The interplay of cell patterning and matrix concentration has high impact on the phenotype and viability of the printed cells. To advance the capabilities of cartilage bioprinting, we investigated different ECMs to create an in vitro chondrocyte niche. Therefore, we used methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA) in a stereolithographic bioprinting approach. Both materials have been shown to support cartilage ECM formation and recovery of chondrocyte phenotype. We used these materials as bioinks to create cartilage models with varying chondrocyte densities. The models maintained shape, viability, and homogenous cell distribution over 14 days in culture. Chondrogenic differentiation was demonstrated by cartilage‐typical proteoglycan and type II collagen deposition and gene expression (COL2A1, ACAN) after 14 days of culture. The differentiation pattern was influenced by cell density. A high cell density print (25 × 106 cells/mL) led to enhanced cartilage‐typical zonal segmentation compared to cultures with lower cell density (5 × 106 cells/mL). Compared to HAMA, GelMA resulted in a higher expression of COL1A1, typical for a more premature chondrocyte phenotype. Both bioinks are feasible for printing in vitro cartilage with varying differentiation patterns and ECM organization depending on starting cell density and chosen bioink. The presented technique could find application in the creation of cartilage models and in the treatment of articular cartilage defects using autologous material and adjusting the bioprinted constructs size and shape to the patient. © 2019 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2649–2657, 2019.

No abstract

These results suggest that SF contains chemokines that may contribute to the recruitment of human mesenchymal progenitors from the subchondral bone in microfracture.