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Photo-Cross-Linkable hydrogel has attracted immense interest in the regeneration of bone repair and regeneration strategies due to its superior biocompatibility and tunable mechanical properties. Recently, Nb was reported to strongly promote the bone regeneration process via an accelerated osteoblast-modulated alkaline phosphatase activity mechanism. In particular, Nb₂C MXenes have drawn widespread attention due to their excellent biocompatibility and ability to induce bone formation. However, the easy agglomeration of Nb₂C nanosheets and subsequent low cell endocytosis efficacy greatly suppressed the osteogenesis effect. In this study, a subtractive nanopore-engineered Nb₂C MXene was prepared through a microwave combustion method, gelatin methacrylate was used as the carrier hydrogel, and the photo-triggered Porous-Nb₂C@GelMA hydrogel was fabricated by a photo-triggered process. The pore-forming strategy not only successfully improved the distribution of Nb₂C and formed more homogenous Porous-Nb₂C@GelMA hydrogels but also guided bone marrow mesenchymal stem cells (BMSCs) toward osteoblast differentiation. Porous Nb₂C provided convenient cellular grasping and endocytosis for BMSCs, which further created a favorable environment for differentiation and osteogenesis. This, in turn, leads to an increase in the expression of osteogenic markers, such as ALP and ARS, as well as osteogenic factors, such as BMP-2, COL-1, OCN, and OPN. Consequently, enhancing the regenerative microenvironment by incorporating porous Nb₂C composite hydrogels shows promise for application in bone regeneration.

The purpose of this investigation was to prepare and evaluate the feasibility and biocompatibility of a new composite as a large defect bone substitute. The new GTGG was mainly composed of tricalcium phosphate ceramic particles and glutaraldehyde crosslinked gelatin in which Gui-Lu-Jiao was added (a mixture of Cervi Colla Cornus and Colla Plastri Testudinis). In the in vitro study, rat's calvaria osteoblasts were used to study bone characteristics upon exposure to different concentrations of the Gui-Lu-Jiao solution. In the in vivo study, GTGG composites were implanted into the defects of calvarial bones in mature New Zealand rabbits to test their osteogenerative characteristics. As a result, we found that Gui-Lu-Jiao added to the culture could promote the proliferation of osteoblasts. In addition, GTGG could induce a large amount of new bone growth in the rabbit's calvarial bone defect. Therefore, the GTGG composite might be a potential bone substitute.

The three-dimensional (3D) nano scaffold of the cellulose acetate (CA) containing graphene/cobalt nanocomposite (0.1wt.%) was fabricated via electrospinning technique, and its impact on bone regeneration was investigated. Through this aim, bone marrow mesenchymal stem cells are cultured on the CA, and graphene/cobalt (rGO/Co)/CA nanocomposite scaffold surfaces and the samples are treated under low frequency alternative magnetic field (75Hz). The scaffolds are characterized using Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and thermal studies (TG/DSC). The proliferation behavior of stem cells on CA, and rGO/Co/CA nano scaffolds are studied by MTT assay, show their biocompability after 14 days of cell seeding. The 6-diamidino-2-phenylindole (DAPI) staining is used to confirm the morality of stem cell for duration of seven days. The nanocomposite scaffold is enhanced for extremely higher proliferation compared to the bare CA scaffold. The acceleration on osteogenic differentiation on the bone mesenchymal stem cell is enhanced within 48h when rGO/Co/CA scaffold is placed under alternative current magnetic field (ACMF). Furthermore, the acceleration of the stem cells differentiation for the rGO/Co/CA scaffold under ACMF corresponds to the induced scaffold surface roughness caused by graphene sheets, the metallic behavior of graphene and the responding of the nanocomposite magnetic parts (i.e., cobalt nanoparticles) while applying 75Hz frequency. Using reverse transcription polymerase chain reaction (RT-PCR) analysis, the superior effect of ACMF on scaffold contain magnetic graphene nanocomposite is confirmed to produce bone related genes within 14 days.

Two kinds of chitosan/β-TCP microspheres were prepared; one was by a traditional emulsion technique (Group A), and the other was by a high-voltage electrostatic system (Group B). Both of the microspheres exhibited good sphericity and the β-TCP fine particles were well trapped inside the chitosan based particles. After 60-day shaking, Group A and Group B degraded by about 40% and 80% of initial weight, respectively. Two models of bone defects were created in rabbits included for a series of randomized blind pilot study. In the cranial model, two equal 10 mm diameter cranial defects were created. In condyle model, two equal 5 mm femur condyle defects were created on each hind leg, and underwent the same grafting treatment. After 4 weeks of implantation, both the cranial and condyle sites filled with Group A were shown to be surrounded by fibrous tissues with the presence of osteoblasts. In Group B, only condyle site showed the presence of osteoblasts. In contrast, fibrous tissue formation was seen on the control group after 4 weeks of healing. After 8 weeks of implantation, the condyle sites filled with Group A and Group B showed the presence of new bone formation as compared to control group. However, there was no obvious new bone formations in the cranial sites filled with both Group A and Group B. The same events were observed in the cranial sites after 12 weeks of implantation. In contrast, new bone formation was seen on the condyle sites in all three groups after 12 weeks of implantation.

In this chapter, bone marrow harvest and isolation as well as mesenchymal stem cell (MSC) culture are described, including sources from human, rabbit, and rat. The biological activity changes of MSCs in the pathogenesis of bone disorders are exemplified by the steroid-associated osteonecrosis rabbit model. As a cell model, the value of MSCs in screening candidate agents against bone disorders is exemplified by icariin, a single-molecule component purified from epimedium, a famous bone-tonifying herb in traditional Chinese medicine. Besides their role as a cell model for the underlying mechanisms, exploration, and corresponding molecular drug screening of bone disorders, MSCs are also a very important source of cell seeds for bone-related tissue engineering repair, which needs a large scale of stem cells. We therefore introduce the concept of MSC in vitro expansion in microcarrier by bioreactor, and the magnetic resonance imaging (MRI) dynamic tracking technique after superparamagnetic iron oxide (SPIO) particle labeling after in vivo transplantation.

In order to understand the mechanisms of fracture healing, especially the neovascularization of the callus, we have established a closed femoral fracture model in rats. This chapter describes a microangiography technique that has been adopted to investigate temporal changes in the three-dimensional (3D) vasculature of the healing callus. Quantitative evaluation protocols for vessel size distribution, total vessel volume, and volume fraction have also been established for comparative studies.

The bone-to-tendon (B-T) or osteotendinous junction is a unique structure within the musculoskeletal system that connects both bone and tendon through the transitional fibrocartilage zone. Injuries to the B-T junction occur as a result of trauma, sports injury, or local chronic inflammation. B-T repair is, however, inferior compared to repair taking place within homogeneous tissues such as bone fracture or tendon repair, as B-T repair involves regeneration of the transitional fibrocartilage zone. Delay in B-T junction healing may often occur, thus preventing early mobilization and rehabilitation. How to accelerate B-T healing is challenging. This chapter describes an experimental partial patellectomy model established for studying both normal and delayed B-T junction healing, with the aim of providing a platform in order to evaluate potential biological and biophysical interventions developed or to be developed for acceleration and/or enhancement of B-T junction repair.

Callotasis or distraction osteogenesis is a well-established orthopedic treatment method in the correction of limb length inequality or bone defect. Plain radiography is commonly used to monitor the progress of distraction osteogenesis, with the known limitation that it can only provide qualitative measurements. Dual-energy X-ray absorptiometry (DXA) is well accepted as the standard clinical equipment for studying bone mineral density in osteoporosis quantitatively with reasonable precision. It has also been successfully used to quantify and monitor bone regeneration in callus distraction, and is able to provide valuable information for clinicians in determining the distraction rate and the timing for frame removal. This chapter summarizes the essential technical details of DXA measurement of the bone mineralization status of callus distraction osteogenesis and its clinical indications.

We prepared zinc-containing TCP/HAp ceramics (Zn-TCP/HAp) in the shape of disk, and examined the effect of zinc on the osteogenic differentiation of cultured marrow cells on the surface of Zn TCP/HAp. Fresh marrow cells were obtained from the femora of Fischer rats and cultured in a medium containing 15% fetal bovine serum to reach confluent. After trypsinization, the cells were seeded at 20×10³ cells/16 mm ϕ on Falcon tissue wells with the ceramic disk for subculture. Just before the cell seeding, three different disks (TCP/HAp containing 0, 0.126, or 0.316 wt% Zn) were placed in the wells. After 2 weeks of subculture in the presence of β-glycerophosphate, vitamin C phosphate, and dexamethasone, the cells were stained for alkaline phosphatase (ALP). The ALP positive area on the Zn-TCP/HAp disk was expanded with an increase in Zn content, as covering almost the entire surface of the Zn-TCP/HAp disk of 0.316 Zn wt%. The results indicate that the Zn doped TCP/HAp ceramics stimulate osteoblastic differentiation in cultured marrow stromal cells.

Effects of porous hydroxyapatite granules (HAGs) on osteogenesis were examined in vitro using cells from adult rabbit femurs. Cultures in the space of 30-600 μm between glass plates with two-dimensionally arranged HAGs on the horizontal bottom glass surface induced thin insular tissue formation within 2 weeks. It was induced earlier and more than that without HAGs. Von Kossa staining-positive tissues were found at 1-2 weeks after their formation, suggesting their mineralization. No cells or several cells in monolayer were included within some of them. Several grew quadrate tissues. Similar thin tissues were formed in inclined and rotated glass tubes with three-dimensionally arranged HAGs. They were almost irregular in shape. By contrast, little extracellular matrix deposition and no mineralization were seen in the spaces over 600 μm and under 30 μm between the glasses with HAGs. These results suggest that osteoblastic cells from adult femurs form mineralized quadrate tissues with or without embedded cells on the horizontal glass in the space of 30-600 μm, while mechanical loading without system induces irregular-shaped tissue formation. Porous HAGs in the space seem to accelerate extracellular matrix accumulation and its mineralization.

A composite of marrow mesenchymal stem cells and porous hydroxyapatite (HA) has in vivo osteogenic potential. To investigate factors enhancing the osteogenic potential of marrow/HA composites, we used recombinant human bone morphogenetic protein-2 (rhBMP-2). Marrow/HA composites or composites containing marrow mesenchymal stem cells, rhBMP-2, and HA (marrow/BMP/HA composites) were implanted subcutaneously in 7-week-old male Fischer rats. BMP/HA composites and HA alone were also implanted. The implants were harvested after 2 and 4 weeks. Histologically, obvious de novo bone formation together with active osteoblasts was seen at both 2 and 4 weeks in many pores of the marrow/BMP/HA composites. The marrow/HA composites did not induce bone formation at 2 weeks, but showed moderate bone formation at 4 weeks. In contrast, neither the BMP/HA composites nor HA alone induced bone formation at any time after implantation. These results indicate that the combination of marrow mesenchymal stem cells, porous HA, and BMP synergistically enhances osteogenic potential and that the composites can be utilized effectively in osseous reconstructive surgery.

Osteopontin(OPN), one of the major non-collagenous bone proteins, has an important role in bone remodeling. OPN not only has a role in triggering the osteoblast early differentiation, but activates osteoclast resorption function. On this point of view, we proposed a model for bone remodeling via integrin-OPN binding, by which OPN could act as a signaling molecule between osteoblasts and osteoclasts in the remodeling procedure. Moreover, OPN proved to induce the in vivo osteogenic potential of bone marrow derived osteoblasts/hydroxyapatitc(HA) construct.