An integral approach toward in situ tissue engineering through scaffolds that mimic tissue with regard to both tissue architecture and biochemical composition is presented. Monolithic osteochondral and meniscus scaffolds are prepared with tissue analog layered biochemical composition and perpendicularly oriented continuous micropores by a newly developed cryostructuring technology. These scaffolds enable rapid cell ingrowth and induce zonal-specific matrix synthesis of human multipotent mesenchymal stromal cells solely through their design without the need for supplementation of soluble factors such as growth factors.

Introduction: Localized trauma-derived breakdown of the hyaline articular cartilage may progress toward osteoarthritis, a degenerative condition characterized by total loss of articular cartilage and joint function. Tissue engineering technologies encompass several promising approaches with high therapeutic potential for the treatment of these focal defects. However, most of the research in tissue engineering is focused on potential materials and structural cues, while little attention is directed to the most appropriate source of cells endowing these materials. In this study, using human amniotic membrane (HAM) as scaffold, we defined a novel static in vitro model for cartilage repair. In combination with HAM, four different cell types, human chondrocytes, human bone marrow-derived mesenchymal stromal cells (hBMSCs), human amniotic epithelial cells, and human amniotic mesenchymal stromal cells (hAMSCs) were assessed determining their therapeutic potential. Material and methods: A chondral lesion was drilled in human cartilage biopsies simulating a focal defect. A pellet of different cell types was implanted inside the lesion and covered with HAM. The biopsies were maintained for 8 weeks in culture. Chondrogenic differentiation in the defect was analyzed by histology and immunohistochemistry. Results: HAM scaffold showed good integration and adhesion to the native cartilage in all groups. Although all cell types showed the capacity of filling the focal defect, hBMSCs and hAMSCs demonstrated higher levels of new matrix synthesis. However, only the hAMSCs-containing group presented a significant cytoplasmic content of type II collagen when compared with chondrocytes. More collagen type I was identified in the new synthesized tissue of hBMSCs. In accordance, hBMSCs and hAMSCs showed better International Cartilage Research Society scoring although without statistical significance. Conclusion: HAM is a useful material for articular cartilage repair in vitro when used as scaffold. In combination with hAMSCs, HAM showed better potential for cartilage repair with similar reparation capacity than chondrocytes.

The aim of this study was to assess a novel approach to treating severe knee osteoarthritis by targeting synovial membrane, superficial articular cartilage, synovial fluid, and subchondral bone by combining intra-articular injections and intraosseous infiltrations of platelet rich plasma. We explored a new strategy consisting of intraosseous infiltrations of platelet rich plasma into the subchondral bone in combination with the conventional intra-articular injection in order to tackle several knee joint tissues simultaneously. We assessed the clinical outcomes through osteoarthritis outcome score (KOOS) and the inflammatory response by quantifying mesenchymal stem cells in synovial fluid. There was a significant pain reduction in the KOOS from baseline (61.55 +/- 14.11) to week 24 (74.60 +/- 19.19), after treatment (p = 0.008), in the secondary outcomes (symptoms, p = 0.004; ADL, p = 0.022; sport/rec., p = 0.017; QOL, p = 0.012), as well as VAS score (p < 0.001) and Lequesne Index (p = 0.008). The presence of mesenchymal stem cells in synovial fluid and colony-forming cells one week after treatment decreased substantially from 7.98 +/- 8.21 MSC/mu L to 4.04 +/- 5.36 MSC/mu L (p = 0.019) and from 601.75 +/- 312.30 to 139.19 +/- 123.61 (p = 0.012), respectively. Intra-articular injections combined with intraosseous infiltrations of platelet rich plasma reduce pain and mesenchymal stem cells in synovial fluid, besides significantly improving knee joint function in patients with severe knee osteoarthritis. This trial is registered on EudraCT with the number 2013-003982-32.

Fracture nonunion is a major complication of bone fracture regeneration and repair. The molecular mechanisms that result in fracture nonunion appearance are not fully determined. We hypothesized that fracture nonunion results from the failure of hypoxia and hematoma, the primary signals in response to bone injury, to trigger Bmp2 expression by mesenchymal progenitor cells (MSCs). Using a model of nonstabilized fracture healing in transgenic 5'Bmp2BAC mice we determined that Bmp2 expression appears in close association with hypoxic tissue and hematoma during the early phases of fracture healing. In addition, BMP2 expression is induced when human periosteum explants are exposed to hypoxia ex vivo. Transient interference of hypoxia signaling in vivo with PX-12, a thioredoxin inhibitor, results in reduced Bmp2 expression, impaired fracture callus formation and atrophic-like nonunion by a HIF-1 alpha independent mechanism. In isolated human periosteum-derived MSCs, BMP2 expression could be induced with the addition of platelets concentrate lysate but not with hypoxia treatment, confirming HIF-1 alpha-independent BMP2 expression. Interestingly, in isolated human periosteum-derived mesenchymal progenitor cells, inhibition of BMP2 expression by PX-12 is accomplished only under hypoxic conditions seemingly through dis-regulation of reactive oxygen species (ROS) levels. In conclusion, we provide evidence of a molecular mechanism of hypoxia-dependent BMP2 expression in MSCs where interference with ROS homeostasis specifies fracture nonunion-like appearance in vivo through inhibition of Bmp2 expression.

The aim of this study was to evaluate the effect of intra-articular (IA) or a combination of intra-articular and intraosseous (IO) infiltration of Platelet Rich Plasma (PRP) on the cellular content of synovial fluid (SF) of osteoarthritic patients. Thirty-one patients received a single infiltration of PRP either in the IA space (n=14) or in the IA space together with two IO infiltrations, one in the medial femoral condyle and one in the tibial plateau (n=17). SF was collected before and after one week of the infiltration. The presence in the SF of mesenchymal stem cells (MSCs), monocytes, and lymphocytes was determined and quantified by flow cytometry. The number and identity of the MSCs were further confirmed by colony-forming and differentiation assays. PRP infiltration into the subchondral bone (SB) and the IA space induced a reduction in the population of MSCs in the SF. This reduction in MSCs was further confirmed by colony-forming (CFU-F) assay. On the contrary, IA infiltration alone did not cause variations in any of the cellular populations by flow cytometry or CFU-F assay. The SF of osteoarthritic patients contains a population of MSCs that can be modulated by PRP infiltration of the SB compartment.