Convergence analysis and validation of a discrete element model of the human lumbar spine

Degenerative diseases of the spine can lead to or hasten the onset of additional spinal problems that significantly reduce human mobility. The spine consists of vertebral bodies and intervertebral discs. The most degraded are intervertebral discs. The vertebral body consists of a shell (cortical bone tissue) and an internal content (cancellous bone tissue). The intervertebral disc is a complex structural element of the spine, consisting of the nucleus pulposus, annulus fibrosus, and cartilaginous plates. To develop numerical models for the vertebral body and intervertebral disc, first, it is necessary to verify and validate the models for the constituent elements of the lumbar spine. This paper, for the first time, presents discrete elements-based numerical models for the constituent parts of the lumbar spine, and their verification and validation. The models are validated using uniaxial compression experiments available in the literature. The model predictions are in good qualitative and quantitative agreement with the data of those experiments. The loading rate sensitivity analysis revealed that fluid-saturated porous materials are highly sensitive to loading rate: a 1000-fold increase in rate leads to the increase in effective stiffness of 130 % for the intervertebral disc, and a 250-fold increase in rate leads to the increase in effective stiffness of 50 % for the vertebral body. The developed model components can be used to create an L4-L5 segment model, which, in the future, will allow investigating the mechanical behavior of the spine under different types of loading.

Immunohistochemical analysis revealed the expression of bone morphogenetic proteins-4, 6, 7, and 9 in human induced membrane samples treated with the Masquelet technique

Background Induced membrane (IM) is the key component of Masquelet reconstruction surgery for the treatment of bone defects. IM is formed around the cement spacer and is known to secrete growth factors and osteoinductive factors. However, there is limited evidence available concerning the presence of osteoinductive factors in IM. This study aimed to investigate the existence of bone morphogenetic proteins (BMPs) in IM harvested from patients during the treatment of bone defects using the Masquelet technique. Methods This study involved six patients whose bone defects had been treated using the Masquelet technique. The affected sites were the femur (n = 3) and the tibia (n = 3). During the second-stage surgery, 1 cm2 pieces of IM were harvested. Histological sections of IM were immunostained with anti-BMP-4, 6, 7, and 9 antibodies. Human bone tissue served as the positive control. Results The presence of BMP-4, 6, 7, and 9 was observed in all IM samples. Further, immunolocalization of BMP-4, 6, 7, and 9 was observed in blood vessels and fibroblasts in all IM samples. Immunolocalization of BMP-4, 6, 7, and 9 was also observed in bone tissue within the IM in one sample, in which osteogenesis inside the IM was observed. Conclusions This study showed that osteoinductive factors BMP-4, 6, 7, and 9 were present in the IM harvested from patients, providing evidence indicating that the Masquelet technique effectively contributes to healing large bone defects. Therefore, it may be possible for surgeons to omit the addition of BMPs to bone grafts, given the endogenous secretion of BMPs from the IM.

Protocol of Co-Culture of Human Osteoblasts and Osteoclasts to Test Biomaterials for Bone Tissue Engineering

New biomaterials and scaffolds for bone tissue engineering (BTE) applications require to be tested in a bone microenvironment reliable model. On this assumption, the in vitro laboratory protocols with bone cells represent worthy experimental systems improving our knowledge about bone homeostasis, reducing the costs of experimentation. To this day, several models of the bone microenvironment are reported in the literature, but few delineate a protocol for testing new biomaterials using bone cells. Herein we propose a clear protocol to set up an indirect co-culture system of human-derived osteoblasts and osteoclast precursors, providing well-defined criteria such as the cell seeding density, cell:cell ratio, the culture medium, and the proofs of differentiation. The material to be tested may be easily introduced in the system and the cell response analyzed. The physical separation of osteoblasts and osteoclasts allows distinguishing the effects of the material onto the two cell types and to evaluate the correlation between material and cell behavior, cell morphology, and adhesion. The whole protocol requires about 4 to 6 weeks with an intermediate level of expertise. The system is an in vitro model of the bone remodeling system useful in testing innovative materials for bone regeneration, and potentially exploitable in different application fields. The use of human primary cells represents a close replica of the bone cell cooperation in vivo and may be employed as a feasible system to test materials and scaffolds for bone substitution and regeneration.

Biomimetic porous scaffolds containing decellularized small intestinal submucosa and Sr2+/Fe3+ co-doped hydroxyapatite accelerate angiogenesis/osteogenesis for bone regeneration

The design of bone scaffolds is predominately aimed to well reproduce the natural bony environment by imitating the architecture/composition of host bone. Such biomimetic biomaterials are gaining increasing attention and acknowledged quite promising for bone tissue engineering. Herein, novel biomimetic bone scaffolds containing decellularized small intestinal submucosa matrix (SIS-ECM) and Sr2+/Fe3+ co-doped hydroxyapatite (SrFeHA) are fabricated for the first time by the sophisticated self-assembled mineralization procedure, followed by cross-linking and lyophilization post-treatments. The results indicate the constructed SIS/SrFeHA scaffolds are characterized by highly porous structures, rough microsurface and improved mechanical strength, as well as efficient releasing of bioactive Sr2+/Fe3+ and ECM components. These favorable physico-chemical properties endow SIS/SrFeHA scaffolds with an architectural/componential biomimetic bony environment which appears to be highly beneficial for inducing angiogenesis/osteogenesis both in vitro and in vivo. In particular, the cellular functionality and bioactivity of endotheliocytes/osteoblasts are significantly enhanced by SIS/SrFeHA scaffolds, and the cranial defects model further verifies the potent ability of SIS/SrFeHA to accelerate in vivo vascularization and bone regeneration following implantation. In this view these results highlight the considerable angiogenesis/osteogenesis potential of biomimetic porous SIS/SrFeHA scaffolds for inducing bone regeneration and thus may afford a new promising alternative for bone tissue engineering.

Changes in the Serum Levels of Estradiol and Changes in Expression of Estrogen Receptor Alpha in the Bone Tissue of Ovariectomized Wistar Rats

Estradiol is an estrogen steroid hormone and is produced basically within the follicles of the ovaries. The decrease in serum estrogens concentration at menopause disrupts the metabolic balance, changes the lipid profile leading to visceral obesity, which caused an increase in serum estradiol levels, through aromatase activity. Estrogen deficiency also is a reason for the development of osteoporosis.We investigated the serum estradiol levels and changes in bone alpha estrogen receptor expression in ovariectomized rats. For this purpose, we used 20 female Wistar rats at reproductive age - 2 months divided into 2 groups: group 1 (G1)-10 animals were ovariectomized and group 2 (G2)-10 of which were sham-operated. All animals of G1 showed weight gain compared to group G2. The results showed that the values of serum 17β-estradiol in rats of G1 statistically increased compared to G2 (p <0.05). Immunohistochemical analysis revealed no difference in estrogen receptor expression between the both groups. Histomorphological analysis of femur from G1 showed the presence of pronounced osteoporosis. Ovariectomy led to the development of obesity, which caused an increase in serum estradiol levels, through aromatase activity, but this process did not prevent bone tissue from developing osteoporosis.

BMP3 Affects Cortical and Trabecular Long Bone Development in Mice

Bone morphogenetic proteins (BMPs) have a major role in tissue development. BMP3 is synthesized in osteocytes and mature osteoblasts and has an antagonistic effect on other BMPs in bone tissue. The main aim of this study was to fully characterize cortical bone and trabecular bone of long bones in both male and female Bmp3−/− mice. To investigate the effect of Bmp3 from birth to maturity, we compared Bmp3−/− mice with wild-type littermates at the following stages of postnatal development: 1 day (P0), 2 weeks (P14), 8 weeks and 16 weeks of age. Bmp3 deletion was confirmed using X-gal staining in P0 animals. Cartilage and bone tissue were examined in P14 animals using Alcian Blue/Alizarin Red staining. Detailed long bone analysis was performed in 8-week-old and 16-week-old animals using micro-CT. The Bmp3 reporter signal was localized in bone tissue, hair follicles, and lungs. Bone mineralization at 2 weeks of age was increased in long bones of Bmp3−/− mice. Bmp3 deletion was shown to affect the skeleton until adulthood, where increased cortical and trabecular bone parameters were found in young and adult mice of both sexes, while delayed mineralization of the epiphyseal growth plate was found in adult Bmp3−/− mice.

The Ability and Mechanism of nHAC/CGF in Promoting Osteogenesis and Repairing Mandibular Defects

Nano-hydroxyapatite/collagen (nHAC) is a new type of bone tissue engineering scaffold material. To speed up the new bone formation of nHAC, this study used concentrated growth factor (CGF) and nHAC in combination to repair rabbit mandibular defects. nHAC/CGF and nHAC were implanted into rabbit mandibles, and X-ray, Micro-CT, HE and Masson staining, immunohistochemical staining and biomechanical testing were performed at 8, 16 and 24 weeks after surgery. The results showed that as the material degraded, the rate of new bone formation in the nHAC/CGF group was better than that in the nHAC group. The results of the HE and Masson staining showed that the bone continuity or maturity of the nHAC/CGF group was better than that of the nHAC group. Immunohistochemical staining showed that OCN expression gradually increased with time. The nHAC/CGF group showed significantly higher BMP2 than the nHAC group at 8 weeks and the difference gradually decreased with time. The biomechanical test showed that the compressive strength and elastic modulus of the nHAC/CGF group were higher than those of the nHAC group. The results suggest that nHAC/CGF materials can promote new bone formation, providing new ideas for the application of bone tissue engineering scaffold materials in oral clinics.