(M-498) In Vivo Evaluation of an Engineered Ceramic Xerogel for Bone-Cartilage Interface Engineering

Introduction:: Osteochondral (OC) defect repair remains a significant clinical challenge due to specific biophysical requirements for grafts to allow for regeneration of both cartilage and bone tissue types present. Existing clinical and tissue engineering methods for OC defect repair often result in poor outcomes [1]. The current repair strategies typically use load-bearing biomaterials or hydrogels in the scaffold design. While biodegradable polymers and ceramics are mechanically stable and provide an osteogenic environment, they have limited water absorption capacity. Conversely, hydrogels mimic native ECM structure and present tissue-like environment, but they do not have the mechanical strength required for load-bearing tissue engineering. To overcome this challenge, a ceramic xerogel with unique properties has been proposed and developed in this study. It was hypothesized that an amorphous silica fiber graft with water of hydration and mechanical characteristics needed can be designed for OC interface engineering. The goal of this study is to develop a novel biomaterial graft and assess its ability to support bone and cartilage layer formation in vivo.

Materials and Methods:: Pure amorphous silica fibers were homogenized in water, block cast, and sintered at 1,550 ℃ to form amorphous silica fiber scaffolds [2]. The resulting porous structures were cooled and cut into 10 mm X 10 mm X 3 mm wafers. Silica fiber matrices used in biocompatibility and osteogenic potential analysis were mineralized with calcium apatite by immersing in 50 mL of 1.5X simulated body fluid (SBF). Pre-mineralized silica scaffolds were then implanted into rats subcutaneously and were evaluated after 6 weeks. Scaffolds used in osteochondral defect implantation were mechanically loaded with primary rabbit chondrocytes spheroids. Prior to infusion, chondrocyte spheroids were created by seeding 10,000 chondrocytes into rounded 2 mL tubes and cultured under mechanical loading through centrifugation at 2,500 gs for 30 minutes daily for 14 days. The mechanically primed spheroids were allowed for fusion to form into lager micro-masses and then encapsulated in a hyaluronic acid hydrogel and loaded onto the silica scaffold. The mechanically primed spheroid grafts were then implanted into a rabbit OC defect model and evaluated after 6 weeks for OC defect repair and regeneration.

Results, Conclusions, and Discussions:: Water absorption studies showed that the silica scaffold can absorb water approximately 500% of their mass (Figure 1A). However, unlike hydrogels the scaffolds did not swell or change morphologically in contact with water, which allows them to maintain their physical structure once implanted into an OC defect [2]. In order to improve the bone regenerative properties, the silica scaffolds were mineralized prior to implantation. To determine the cell compatibility of the unmineralized and pre-mineralized amorphous silica scaffolds, the samples were seeded with primary hMSCs and cultured in vitro. Subsequent Live/Dead staining showed significant growth and proliferation of the cells for each scaffold (Figure 1B). Sintering silica fibers at 1,550 °C revealed complete sintering of adjoining silica fibers, forming large pore spaces whose average pore size fits within the size range needed for cell infiltration and nutrient transport (50-300 um) [2]. After 21 days of biomineralization, the surface of the silica scaffolds was covered with precipitates that nucleated to large macroscale structures. Unmineralized silica scaffolds that were loaded with mechanically primed chondrocyte spheroids displayed increased chondrogenic activity during in vitro culture (Data not shown). 6 weeks post-implantation in a rabbit osteochondral model, histological evaluation showed that the mechanically loaded spheroids formed a dense cell-rich layer in the articular cartilage area of the defect (Figure 2C). Safranin O staining demonstrated that the mechanically loaded spheroids were developed a cartilage-like matrix rich in glycosaminoglycans. Pre-mineralized silica scaffolds were implanted subcutaneously to evaluate their biocompatibility and osteogenic potential (Figure 2D). 6 weeks post-implantation, Hematoxylin and Eosin (H&E) staining showed that pre-mineralized scaffolds developed minimal immune response with negligible fibrous encapsulation, similar to that of PLGA, therefore proving the biocompatibility of the newly developed ceramic xerogel. Masson’s Trichrome (MT) staining showed bule regions within the scaffold pore structure indicating the development of collagen fibers and osteogenesis, which is significant since the scaffold alone was implanted subcutaneously. Overall, the results demonstrate that the newly developed ceramic xerogel can support both bone and cartilage-layer formation individually. Further studies are in progress to evaluate the selectively mineralized and chondrocyte-loaded graft ability to support bone-cartilage interface engineering.


Acknowledgements (Optional): : National Institute of Biomedical Imaging and Bioengineering (NIBIB) of the National Institutes of Health (#R01EB030060 & #R01EB020640).

References (Optional): : 1. Nukavarapu and Dorcemus, Biotechnology Advances 2013; 31(5):706-721 2. Kim et al., Bioactive Materials 2023; 19:155-166