A Cellularized Biphasic Implant Based on a Bioactive Silk Fibroin Promotes Integration and Tissue Organization during Osteochondral Defect Repair in a Porcine Model

Abstract

In cartilage tissue engineering, biphasic scaffolds (BSs) have been designed not only to influence the recapitulation of the osteochondral architecture but also to take advantage of the healing ability of bone, promoting the implant’s integration with the surrounding tissue and then bone restoration and cartilage regeneration. This study reports the development and characterization of a BS based on the assembly of a cartilage phase constituted by fibroin biofunctionalyzed with a bovine cartilage matrix, cellularized with differentiated autologous pre-chondrocytes and well attached to a bone phase (decellularized bovine bone) to promote cartilage regeneration in a model of joint damage in pigs. BSs were assembled by fibroin crystallization with methanol, and the mechanical features and histological architectures were evaluated. The scaffolds were cellularized and matured for 12 days, then implanted into an osteochondral defect in a porcine model (n = 4). Three treatments were applied per knee: Group I, monophasic cellular scaffold (single chondral phase); group II (BS), cellularized only in the chondral phase; and in order to study the influence of the cellularization of the bone phase, Group III was cellularized in chondral phases and a bone phase, with autologous osteoblasts being included. After 8 weeks of surgery, the integration and regeneration tissues were analyzed via a histology and immunohistochemistry evaluation. The mechanical assessment showed that the acellular BSs reached a Young’s modulus of 805.01 kPa, similar to native cartilage. In vitro biological studies revealed the chondroinductive ability of the BSs, evidenced by an increase in sulfated glycosaminoglycans and type II collagen, both secreted by the chondrocytes cultured on the scaffold during 28 days. No evidence of adverse or inflammatory reactions was observed in the in vivo trial; however, in Group I, the defects were not reconstructed. In Groups II and III, a good integration of the implant with the surrounding tissue was observed. Defects in group II were fulfilled via hyaline cartilage and normal bone. Group III defects showed fibrous repair tissue. In conclusion, our findings demonstrated the efficacy of a biphasic and bioactive scaffold based on silk fibroin and cellularized only in the chondral phase, which entwined chondroinductive features and a biomechanical capability with an appropriate integration with the surrounding tissue, representing a promising alternative for osteochondral tissue-engineering applications.

Host Response to Biomaterials for Cartilage Tissue Engineering: Key to Remodeling

“…The fabrication of functionalized scaffolds with biological activity made of natural biomaterials is undoubtedly a promising approach ( Feng et al, 2020 ; Vainieri et al, 2020 ). Natural biomaterials such as dECM and natural polymers (collagen, silk fibroin, alginate, and chitosan) show repair potential in animal models of cartilage lesion ( Dai et al, 2019 ; Pérez-Silos et al, 2019 ; Singh et al, 2019 ). Tissue (or organ) from various species after decellularization can be utilized for the repair of a damaged tissue ( Sun et al, 2018 ; Lindberg et al, 2019 ).…”

Sequential growth factor exposure of human Ad‑MSCs improves chondrogenic differentiation in an osteochondral biphasic implant

“…In the present study, the biphasic scaffolds developed had an upper 2-mm thick phase that simulates native cartilage conditions for further implantation, with native cartilage characteristics, such as flexibility, water retention and mechanical resistance to compression ( 25 ). The lower 4-mm thick phase of decellularized bone matrix chips served as support for the cartilage phase implantation and integration of cartilage with adjacent tissue.…”

“…Full decellularization was assured by H&E staining. Bovine bone chips were obtained by perforation of the same bovine knees after cross-sectional slicing of the condyle area, followed by decellularization with hydrogen peroxide according to the decellularization method described by Pérez-Silos et al ( 25 ) to avoid host rejection of the material ( 26 ).…”

Osteochondral tissue engineering: Perspectives for clinical application and preclinical development

“…In the animal studies with biphasic scaffolds, some studies set a control group in which is a single phasic scaffold was used [ [39] , [40] , [41] , [42] , [43] , [44] , [45] ]. Compared to the hyaline cartilage and porous bone regeneration with a clear tidemark observed in the group pf biphasic scaffolds, the inferior outcomes in the group of single chondral phase scaffolds included: subchondral bone formation and disordered fibrocartilage [ 39 ]; Disorganized fibrous tissue in both bone and chondral phase [ 40 ]; Lower ICRS II scores without regeneration of subchondral bone [ 41 ]; Slower cartilage and bone regeneration and integration with the native tissue [ 42 ]; A mixture of fibrocartilage and cartilage tissue [ 43 ]; Slower regeneration of bone layer [ 44 ]; fibrotic tissue separated from the surrounding bone [ 45 ]. In the studies of triphasic scaffolds, the corresponding control group of monophasic scaffolds also resulted in limited bone and cartilage repair with poor quality [ 34 , 46 ].…”

“…Many positive results have been reported using the combination of chondrocytes in the chondral layer and MSCs or osteoblasts in the bony layer for OCD repair, but several studies showed preloaded cells in the bone layer might lead to fibrocartilage formation due to excessive blood vessels formation. Perez-Silos group [ 40 ] found the scaffolds in which both osteoblasts and chondrocytes were loaded in the bone layer and chondral layer respectively (group 3) resulted in fibrocartilage repair. However, the group in which only chondrocytes were loaded on to the chondral layer (group 2) showed organized collagen II fibers and proteoglycans at the chondral layer and trabeculae in the bone layers.…”