Osteochondral Tissue Engineering In Vivo: A Comparative Study Using Layered Silk Fibroin Scaffolds from Mulberry and Nonmulberry Silkworms

Abstract

The ability to treat osteochondral defects is a major clinical need. Existing polymer systems cannot address the simultaneous requirements of regenerating bone and cartilage tissues together. The challenge still lies on how to improve the integration of newly formed tissue with the surrounding tissues and the cartilage-bone interface. This study investigated the potential use of different silk fibroin scaffolds: mulberry (Bombyx mori) and non-mulberry (Antheraea mylitta) for osteochondral regeneration in vitro and in vivo. After 4 to 8 weeks of in vitro culture in chondro- or osteo-inductive media, non-mulberry constructs pre-seeded with human bone marrow stromal cells exhibited prominent areas of the neo tissue containing chondrocyte-like cells, whereas mulberry constructs pre-seeded with human bone marrow stromal cells formed bone-like nodules. In vivo investigation demonstrated neo-osteochondral tissue formed on cell-free multi-layer silk scaffolds absorbed with transforming growth factor beta 3 or recombinant human bone morphogenetic protein-2. Good bio-integration was observed between native and neo-tissue within the osteochondrol defect in patellar grooves of Wistar rats. The in vivo neo-matrix formed comprised of a mixture of collagen and glycosaminoglycans except in mulberry silk without growth factors, where a predominantly collagenous matrix was observed. Immunohistochemical assay showed stronger staining of type I and type II collagen in the constructs of mulberry and non-mulberry scaffolds with growth factors. The study opens up a new avenue of using inter-species silk fibroin blended or multi-layered scaffolds of a combination of mulberry and non-mulberry origin for the regeneration of osteochondral defects.

Potential of non-mulberry silk protein fibroin blended and grafted poly(Є-caprolactone) nanofibrous matrices for in vivo bone regeneration

“…The inherent integrin binding tri-peptide sequence (Arg-GlyAsp) of A. mylitta fibroin [9] serves to augment cellular interactions due to better cell attachment, and thus aid bone regeneration. Earlier studies have shown that both 2D and 3D matrices of fibroin from A. mylitta promote osteogenic differentiation of human MSCs [ 22], while our previous investigations yielded that scaffolds fabricated using 2 wt% non-mulberry NSF are a better choice for cell proliferation [23].Due to only a limited number of in vivo studies having been performed with SF from Antheraea mylitta [14,15], the possibility of translating nanofibrous NSF scaffolds to clinical trial phase remains an uphill task.…”

“…PCL itself is reasonably biocompatible and biodegradable, supports a wide range of cells [20], and can be conveniently electrospun into nanofibrous matrices. Silk, as a raw material for tissue engineering, has abundant availability, is FDA approved for repair of tendons and ligaments, can serve as a regenerative template for several tissues, and is commercially marketed for biomedical applications [14]. Such aspects are not observed for most other biomaterials in contention for tissue engineering applications.…”

“…As a logical advancement of the in vitro trials, in vivo investigations are organized for the NSF blended and grafted matrices. While in vivo studies of silk fibroin have mostly involved silk fibroin taken Bombyx Mori [12,13], only few studies have used silk fibroin from A. mylitta [14,15]. This is in spite of the more favourable characteristics of the NSF from A.mylitta, both in vivo [14,15] and in vitro [8,10].…”

“…While in vivo studies of silk fibroin have mostly involved silk fibroin taken Bombyx Mori [12,13], only few studies have used silk fibroin from A. mylitta [14,15]. This is in spite of the more favourable characteristics of the NSF from A.mylitta, both in vivo [14,15] and in vitro [8,10]. Implanting cell-free scaffolds has certain useful aspects when leading up to clinical trials, like minimal added expenses, absence of complications from the need to isolate cells from patients, and no risk of graft rejection by host body.…”

In vitro and in vivo studies of a novel bacterial cellulose-based acellular bilayer nanocomposite scaffold for the repair of osteochondral defects

“…The total amount of growth factors loaded onto the scaffolds was 1 µg BMP-2 and 5 ng TGF-β3. 28 Intraperitoneally injected mixture of ketamine HCl (80 mg/Kg) and xylazine (10 mg/Kg) served as an anesthetic for animals. OCD (1 mm diameter ×3 mm depth) were created in the patellar groove of the knee joints using both a hand drill and a dental burr (Figure S1B-G).…”