Hydroxyapatite and Loofah-Reinforced Carboxymethyl Chitosan Hydrogel Scaffold with/without Stem Cells for Bone Healing: In Vitro and In Vivo

Abstract Bone tissue is a structure composed of organic and inorganic components. There are some problems associated with the fracture healing of bone tissue. The fracture union and rehabilitation process can take a long time due to infections, delayed healing, or nonunion of the bone tissue. Recent studies have directed orthopedic surgeons to cellular therapy and biomaterials. This study investigated hydroxyapatite (HAp) and loofah-reinforced carboxymethyl chitosan composite scaffolds (HAp + loofah + CMCht) in vitro and in vivo fracture healing. Following material characterizations, in vitro biocompatibility analyses were conducted using rat mesenchymal stem cells derived from periosteum (pMSCs) and bone marrow (bMSCs) tissues. Moreover, in vivo studies were conducted on 48 Wistar Albino rats, randomly divided into 4 groups: empty defect, cell-free scaffold, bMSCs-seeded scaffold, and pMSCs-seeded scaffold. The rats were followed up for 8 weeks after implantation. Biomechanical, radiological, and histological examinations were performed in vivo. Our results showed that the HAp + loofah + CMCht scaffold is biocompatible and an excellent alternative for fracture healing studies with mesenchymal stem cells. ALP activity was measured to evaluate the bone-forming ability of the bMSCs and pMSCs on the scaffolds. bMSCs-seeded scaffolds increased significantly between days 7 and 28 ( p < 0.01). The scaffolds populated with pMSCs showed a slight, non-significant decline by day 21 of incubation. However, a significant increase was noted when comparing days 21 and 28 in the pMSCs-seeded scaffolds ( p < 0.0002). Analysis of cell presence, morphology, distribution, and adhesion in HAp + loofah + CMCht scaffolds indicated enhanced cell proliferation on days 14 and 21 compared to day 7, as demonstrated by histochemical and immunohistochemical assays. The bMSC exhibited superior osteoconductive properties compared to the pMSC, as demonstrated through comprehensive in vitro and in vivo studies. These experiments revealed that the bMSC not only facilitated enhanced bone tissue formation but also promoted faster integration with existing bone structures. The results indicate that the unique characteristics of bMSCs significantly contribute to their effectiveness in supporting bone regeneration, making them a promising candidate for applications in orthopedic and reconstructive medicine.