• biomaterials
• osteochondral defects
• scaffold
• tissue engineering

Acute and degenerative chondral and osteochondral lesions represent a public health problem related to the worldwide increase of the aging population, unhealthy lifestyles and sport injuries that largely lead to disability and to a worsening quality of life. These conditions affect an ever-increasing number of people worldwide with huge socio-economic impacts. Even though several therapeutic approaches have been developed to treat these kinds of defects, none of them has yet proved to be quite successful, as they have drawbacks and limitations and often do not provide sufficient tissue regeneration and recovery of original functions. It is well known that articular cartilage repair does not occur or requires long times, producing unsatisfactory results in term of functional and structural characteristics compared to the original tissues. Thus, there is an increasing demand of new strategies able to promote tissue regeneration and provide a better quality of life. The driving force in these recent years has been represented by tissue engineering strategies in which cells of different origin, scaffolds, bioactive factors and physical stimuli were combined together to grow biological constructs, acting as substitutes that restore, maintain or improve the functionality of damaged tissue and organs. The regeneration of osteochondral defects has been attracting a great deal of attention since it is considered a difficult goal to achieve given the dual composition in which cartilage and subchondral bone with intrinsic biological, biochemical, and biomechanical differences and properties exist. Especially in the regeneration of the osteochondral tissue, the development of the appropriate scaffold architecture that can support either bone and cartilage tissues play a key role. One of the most promising strategies is represented by the realization of a bilayered scaffold, obtained by the association of different and distinct but integrated scaffold layers that mimic cartilage and subchondral bone. In fact, one feasible approach to develop the scaffold for the osteochondral regeneration with suitable biological properties is to follow a biomimetic approach that consists in the development of biologically inspired materials which possess physical and/or chemical properties similar to those of the native tissues.
With respect to the current knowledge, the present project intended to develop new scaffolds for osteochondral tissue regeneration through a “biomimetic” and "bioinspired" approach. Scaffolds were realized with an organic compound (type I collagen) for the chondral regeneration and onto the co-precipitation of the organic component with bioactive Magnesium-doped hydroxyapatite (Mg/HA) crystals for the regeneration of the subchondral layer. Scaffolds were stabilized with a cross-linking agent bis-epoxyde highly reactive (1,4-butanediol diglycidyl ether - BDDGE). Different concentrations of BDDGE (0.05wt%,1wt% and 4wt%) associated with different reaction temperatures (4 °C and 37 °C) and pH values (pH = 7 in milli-Q water and pH = 9.5 in NaHCO3/Na2CO3 buffer solution) were combined in order to identify the optimal crosslinking condition for chondral and bone scaffold layer development. The developed scaffolds were characterized from the physico-chemical point of view investigating their morphological and microstructural properties by scanning electron microscopy (SEM), their enzymatic stability and the crosslinking degree. X-ray diffraction analysis was also performed to investigate the structure of the scaffolds and finally a delamination was carried out onto the integrated chondral and bone scaffold layers into the bilayered osteochondral scaffold, to assess the adhesion strength between the integrated layers. The physico-chemical characterization demonstrated that the crosslinking process significantly improved the properties of both chondral and bone scaffold layers in comparison with uncrosslinked scaffold, playing an essential role in the creation of porosity and stability and that the reactions conditions (temperature and pH) significantly influenced the crosslinking reaction performance. The analysis and comparison of the overall physico-chemical results suggested that the crosslinking reaction is more efficient when performed in carbonate buffer solution and alkaline pH and that BDDGE 1wt% represents the best crosslinking agent concentration to develop a scaffold with suitable properties for osteochondral tissue engineering applications.
The intrinsic cytotoxicity of the chondral and bone scaffold layers was investigated by carrying out a direct contact cytotoxicity assay adopting appropriate biological parameters, as established by the International Standard Organization: UNI EN ISO10993 Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity (2009). Results have shown no cytotoxic effect induced onto fibroblast-like cell line by chondral and bone scaffold layers for 24 and 72 hours. Cells were viable and proliferated in presence of both scaffold layers, with a viability and morphology characteristics comparable to those obtained for the negative control. Complementarily in vitro cell culture studies were carried out to evaluate the capacity of chondral and bone scaffold layers to separately support the growth and differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts and chondrocytes, respectively. To this end at 24 hours, 2, 3 and 4 weeks cell adhesion and morphology were evaluated by scanning electron microscopy (SEM) and histological evaluations while the cell viability and proliferation were assessed by DNA quantification and Live/Dead® staining. The chondrogenic and osteogenic differentiation of hMSC was evaluated measuring the glycosaminoglycan synthesis and alkaline phosphatase activity and by investigating the expression of specific genes of chondrogenic and osteogenic differentiation by RT-PCR analysis. The results showed that chondral and bone scaffold layers represented biocompatible scaffolds able to sustain hMSC attachment and proliferation. In addition, the results indicated that the association of scaffold stimuli and differentiation medium induced the chondrogenic and osteogenic differentiation of hMSCs and the deposition of extracellular matrix (ECM) during the experimental times.
An in vivo implantation study was also performed to screen the inflammatory properties and to evaluate the scaffold osteoinductive/chondroinductive capability. Chondral and bone scaffold layers were combined into an osteochondral bilayered scaffold. Seventeen immunocompromised mice received a subcutaneous implant of osteochondral scaffolds engineered or not with hMSCs. After 4 and 8 weeks, the retrieved samples were histologically evaluated and scored with a histological semiquantitative grading score for soft tissue. At both experimental times the histological evaluation showed no significant inflammatory reactions. Ectopic implantation of scaffolds after seeding with hMSC indicated that cells were able to penetrate deeply into the osteochondral scaffold that appeared permissive to the tissue growth and penetration ensuring the diffusion of nutrients and oxygen, as also suggested by the presence of neo-angiogenesis process especially at 4 weeks. At 8 weeks, a chondrogenic and osteogenic inductive potentials of hMSCs were obtained with the clear formation of bone tissue trabeculae and chondrocyte-like arrangement and microsphere-like formation.
The obtained results, concerning the physicochemical and biological properties (in vitro and in vivo) of the developed bilayered osteochondral scaffolds, demonstrated that the scaffold is not cytotoxic, is histocompatible and it is able to provide an adequate 3D support for the attachment, proliferation and differentiation of hMSCs. In vivo it is able to support the formation of new bone and chondral tissue thus exhibiting a great potential for its use in tissue engineering strategies.