With the ultimate goal to generate suitable materials for the repair of osteochondral defects, in this work we aimed at developing composite osteochondral scaffolds organized in different integrated layers, with features which are biomimetic for articular cartilage and subchondral bone and can differentially support formation of such tissues.A biologically inspired mineralization process was first developed to nucleate Mg-doped hydroxyapatite crystals on type I collagen fibers during their self assembling. The resulting mineral phase was non-stoichiometric and amorphous, resembling chemico-physical features of newly deposited, natural bone matrix. A graded material was then generated, consisting of (i) a lower layer of the developed biomineralized collagen, corresponding to the subchondral bone, (ii) an upper layer of hyaluronic acid-charged collagen, mimicking the cartilaginous region, and (iii) an intermediate layer of the same nature as the biomineralized collagen, but with a lower extent of mineral, resembling the tidemark. The layers were stacked and freeze-dried, to obtain an integrated, monolithic composite. Culture of the material for 2 weeks after loading with articular chondrocytes yielded cartilaginous tissue formation selectively in the upper layer. Conversely, ectopic implantation in nude mice of the material after loading with bone marrow stromal cells resulted in bone formation which remained confined within the lower layer.In conclusion, we developed a composite material with cues which are biomimetic of an osteochondral tissue and with the capacity to differentially support cartilage and bone tissue generation. The results warrant test of the material as a substitute for the repair of osteochondral lesions in orthotopic animal models. Response to Reviewer #1:1) results and discussion should be separated We would prefer to maintain Results and Discussion combined, in order to avoid possible redundancies and improve readability. 2) figures should be detailed with arrows and information about specific orientations. Details and arrows have been included in the Figures3) collagen type 1 was used for the creation of the construct -in every layer? Usually collagen type 1 does not play a role in articular cartilage. This should be addressed in the discussion. According to the Reviewer's comment, a rationale for the use of type I collagen has been included in the text, as follows: "Type I collagen was selected for the synthesis of the composite layers, including the cartilaginous one, due to (i) its good physico-chemical stability and processability and (ii) high safety and biocompatibility profile, related to the removal of all Reply Letter to Editor and referee's comments telopeptides, which are potentially responsible for immunological reactions." (Section 2.1, Page 2, 1 st paragraph). Indeed, type II collagen is normally extracted porcine derivative, and is still not available either in bulk for processing or for clinical use. Nowadays there are only few papers on experimental studies desc...

The objective of this article was to investigate the safety and regenerative potential of a newly developed biomimetic scaffold when applied to osteochondral defects in an animal model. A new multilayer gradient nano-composite scaffold was obtained by nucleating collagen fibrils with hydroxyapatite nanoparticles. In the femoral condyles of 12 sheep, 24 osteochondral lesions were created. Animals were randomized into three treatment groups: scaffold alone, scaffold colonized in vitro with autologous chondrocytes and empty defects. Six months after surgery, the animals were sacrificed and the lesions were histologically evaluated. Histologic and gross evaluation of specimens showed good integration of the chondral surface in all groups except for the control group. Significantly better bone regeneration was observed both in the group receiving the scaffold alone and in the group with scaffold loaded with autologous chondrocytes. No difference in cartilage surface reconstruction and osteochondral defect filling was noted between cell-seeded and cell-free groups. In the control group, no bone or cartilage defect healing occurred, and the defects were filled with fibrous tissue. Quantitative macroscopic and histological score evaluations confirmed the qualitative trends observed. The results of the present study showed that this novel osteochondral scaffold is safe and easy to use, and may represent a suitable matrix to direct and coordinate the process of bone and hyaline-like cartilage regeneration. The comparable regeneration process observed with or without autologous chondrocytes suggests that the main mode of action of the scaffold is based on the recruitment of local cells. ß

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The present in vivo preliminary experiment is aimed at testing mechanical and biological behaviour of a new nano-structured composite multilayer biomimetic scaffold for the treatment of chondral and osteochondral defects. The three-dimensional biomimetic scaffold (Fin-Ceramica Faenza S.p.A., Faenza-Italy) was obtained by nucleating collagen fibrils with hydroxyapatite nanoparticles, in two configurations, bi- and tri-layered, to reproduce, respectively, chondral and osteochondral anatomy. Chondral defects (lateral condyle) and deep osteochondral defects (medial condyle) were made in the distal epiphysis of the third metacarpal bone of both forelimbs of two adult horses and treated respectively with the chondral and osteochondral grafts. Both animals were euthanised six months follow up. The images obtained at the second look arthroscopy evaluation, performed two months after surgery, demonstrated good filling of the chondral and osteo-chondral defects without any inflammatory reaction around and inside the lesions. At the histological analysis the growth of trabecular bone in the osteochondral lesion was evident. Only in one case, the whole thickness of the osteochondral lesion was filled by fibrocartilaginous tissue. The formation of a tidemark line was evident at the interface with the newly formed bone. Newly formed fibrocartilaginous tissue was present in the area of the chondral defect. Initial alignment of the collagen fibres was recognisable with polarised light in both groups. The results of the present pilot study showed that this novel osteochondral and chondral scaffold may act as a suitable matrix to facilitate orderly regeneration of bone and hyaline-like cartilage.

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