| Osteoporosis is a worldwide emerging healthcare issue and socioeconomic threat characterized by low bone mass, poor bone strength and microarchitectural deterioration in trabecular and cortical skeleton, leading to increased bone fragility and susceptibility to fractures. [1] It is an age-related disease caused by the imbalance between osteoblastic bone formation and osteoclastic bone resorption commonly resulting from postmenopausal estrogen deficiency [2]. Over 200 million people are estimated to be affected by osteoporosis worldwide [3], and approximately 50% of 65-year-old postmenopausal white or Asian women will experience an osteoporotic fracture [4]. For decades, several studies have shown that defect healing in postmenopausal osteoporotic women are delayed, mainly due to the absence of estrogen associated with a rise in osteoclast number [15,16]. At present, the two major pharmacological approaches for the treatment of osteoporosis are anabolic agents such as parathyroid hormone (PTH) by stimulating bone formation and anti-resorptive agents including bisphosphonates, calcitonin, raloxifene, and estrogen which act by inhibiting bone resorption [17]. Although much emphasis has been given to the treatment of osteoporosis and fracture prevention by using the above mentioned agents, less investigation has been conducted on the therapeutic effect of local transplantation of biomaterial scaffolds during osteoporotic defect regeneration.Silk is basically composed of fibrous polypeptide in P-sheet form, whose main component is glycine, alanine and sericin [6]. It can be naturally derived from insects and formed via genetic engineering or the modification of native silk fibroin sequence chemistries.[18] Considering the superior properties of lightweight, degradable, high tensile strength, tough, inexpensive and ease of processing [7], it provided new options to further expand the utilization of silk fibroin-based scaffolds for ligament, bone and cartilage tissue regeneration. [8,9] Several properties including less toxic, non-inflammatory, non-immunogenic, biodegradable and bioresorbable were tested by both in vitro and in vivo studies. [8,19,20] Accordingly, they have been gaining widespread attention as controlled-release carriers or templates for cellular activities in bone tissue engineering, whereas the osteoconductivity of pure silk scaffold is inferior compared to ceramic scaffolds. In contrast, ceramic scaffolds such as calcium phosphate (CaP) and bioglass have been the most commonly used biocompatible bone substitute for both dental and orthopedic applications, due to its similar inorganic composition, structure and mechanical properties to the mineralized bone tissue [10]. CaP exhibit a high protein binding strength and the dissolution of inorganic ions is followed by the reprecipitation of mineralized phase between bone tissue and scaffolds, yielding enhanced bone-matrix interface strength [11]. On the other hand, Bioactive glasses (BGs), referred to as the third generation of biomaterials for in situ tissue regeneration, have the capability to bond to both bone and soft tissue [12]. The release of Na+ and Ca2+ ions and the deposition of a carbonated hydroxyapatite layer form a strong chemical bond between glass and host bone, thus stimulate new bone growth [13]. Additionally, a new member of bioactive glasses mesoporous bioglasses (MBGs), elicit more superior bioactivity than BGs, owing to the highly improved surface and porous volume. Both intracellular and extracellular responses were rapidly improved at the interface of the glass that release soluble Si, Ca, P and Na ions, which promote their novel application in biomedical science[14]Thus, we set up three series of experiments according to the problems:(1) we establish rat ovariectomized model and investigate the osteogenesis of mesoporous and non-mesoporous/silk scaffolds in both normal and osteoporotic mesenchymal stem cells, including cell attachment, proliferation and osteogenic differentiation; (2) we compare the osteogenesis of mesoporous and non-mesoporous/silk scaffolds in osteoporotic femur defect model, including bone formation, mineralization, matrix maturation, chondrogenesis and osteoclastic activity, etc.; (3) we also investigate the osteogenesis of CaP/silk scaffolds in the same osteoporotic femur defect model,, including bone formation, mineralization, matrix maturation, chondrogenesis and osteoclastic activity, etc.Part I Comparative study on the osteogenesis of mesoporous and non-mesoporous/silk scaffolds in both normal and osteoporotic mesenchymal stem cellsObjective:The aim of this study is to identify both normal and osteoporotic MSCs behavior in vitro by using 3D BG/silk and MBG/silk scaffolds, and pure silk scaffolds as control.Materials and methods:Rats (weight 200 ± 10 g) were subjected to bilateral ovariectomy (OVX) or sham operation (Sham) at 3 months of age. After 2-month-induction, μCT, HE and Alizarin Red staining were performed to confirm the establishment of osteoporosis model. Mesenchymal stem cells (MSCs) from both OVX and Sham rats were isolated and cultured in 3D BG/silk, MBG/silk and pure silk scaffolds. Cell-Counting Kit (CCK)-8 was used to investigate O-MSC/S-MSC proliferation, pretreated cell-scaffold constructs were observed under SEM for cellular morphology, and Quantitative alkaline phosphatase (ALP) activity was measured to determine the osteogenic differentiation after induction.Results:Compared with sham control, μCT images showed a significant decrease in bone structural parameters such as BV/TV, Tb.N and Tb.Th, but increased Tb.Sp, as well as the reduced cortical thickness. Primary MSCs isolated from osteoporotic rats were confluent after 12 d of culture, while the confluent time of MSCs from their sham littermate was 9d. The results of SEM, CCK-8 assay and quantitative ALP activity showed that MBG/silk scaffolds can improve attachment, proliferation and osteogenic differentiation of both O-MSCs and sham control.Conclusions:These in vitro data suggested that MBG/silk scaffolds provide a better environment for both normal and osteoporotic MSCs attachment, proliferation and osteogenic differentiation. They are promising candidate for in vivo bone defect models.Part II Comparative study on the osteogenesis of mesoporous and non-mesoporous/silk scaffolds in osteoporotic bone defectObjective:This study investigated a biomaterial-based approach for accelerating OVX defect healing by using 3D BG/silk and MBG/silk scaffolds, and pure silk scaffolds as control.Materials and methods:Wistar rats were used to induce postmenopausal osteoporotic model by bilateral ovariectomy. A 2.5-mm diameter latero-lateral bicortical channel was created approximately beneath the growth plate and perpendicular to the shaft axis, The pure silk, BG/silk and MBG/silk scaffolds were placed into these critical sized defects. At time points,2 and 4 weeks after scaffold insertion, rats were sacrificed and all specimens were designated to μCT analysis, the in vivo osteogenetic efficiency was evaluated by μCT analysis, hematoxylin and eosin staining, Safranin O staining, tartrate-resistant acid phosphatase staining, and immunohistochemical assessment (COL I, OPN, BSP and OCN).Results:In vivo results indicate accelerated bone formation with compatible scaffold degradation and reduced osteoclastic response of defect healing in OVX rats after 2 and 4 weeks treatment, with a rank order of MBG/silk> BG/silk> silk group. Immunohistochemical markers of COL I, OPN, BSP and OCN also revealed that MBG/silk scaffolds can better induce accelerated collagen and non-collagen matrix production.Conclusions:The findings of this study suggest that synthesized MBG/silk scaffolds can be used as bone substitutes for local implantation into critical sized osteoporotic defects, owing to and accelerated in vivo healing progress over skeletal deterioration compared with silk and BG/silk scaffolds. The rapid mineralization and reduced osteoclastic activity of MBG/silk scaffolds suggests their potential therapeutic efficacy especially in the site of post-menopausal osteoporosis.Part III The osteogenesis of CaP/silk scaffolds in osteoporotic bone defectObjective:The aim of the present study was to compare the involved remodeling process and therapeutic effect of porous CaP/silk composite scaffolds upon local implantation into osteoporotic defects.Materials and methods:Wistar rats were used to induce postmenopausal osteoporotic model by bilateral ovariectomy. The pure silk and hybrid CaP/silk scaffolds were implanted into critical sized defects created in distal femoral epiphysis. After 14 and 28 days, the in vivo osteogenetic efficiency was evaluated by uCT analysis, hematoxylin and eosin staining, Safranin O staining, tartrate-resistant acid phosphatase staining, and immunohistochemical assessment. Animals with or without critical-sized defects were used as drill or blank controls, respectively.Results:The osteoporotic defect model was well established with significantly decreased 1CT parameters of BV/TV, Tb.N and increased Tb.Sp, porosity, combined with changes in histological observations. During the healing process, the critical-sized drill control defects failed to regenerate appreciable bone tissue, while more significantly increased bone formation and mineralization with dynamic scaffold degradation and decreased osteoclastic bone resorption could be detected within defects with hybrid CaP/silk scaffolds compared to pure silk scaffoldsConclusions:Porous CaP/silk composite scaffolds are more useful by osseointegrating well after local implantation into critical sized osteoporotic defects, compared with pure silk scaffolds as bone substitute biomaterials. The data demonstrates an activated remodeling process both through intramembranous and endochondral bone formation over skeletal deterioration. The accelerated mineralization and dynamic degradation of our scaffolds suggest the potential therapeutic efficacy of hybrid CaP/silk for the initial healing of osteoporotic defects. |
