The intervertebral disc (IVD) has a central nucleus pulposus (NP) able to resist compressive loads and an outer annulus fibrosus which withstands tension and gives mechanical strength. The tissue engineering of a disc substitute represents a challenge from mechanical and biological (nutrition and transport) points of view. Two hyaluronan-derived polymeric substitute materials, HYAFF® 120, an ester and HYADD® 3, an amide were injected into the NP of the lumbar spine of female pigs (11.1 ± 1.0 Kg) in which a nucleotomy had also been performed. Homologous bone marrow stem cells, obtained from the bone marrow three weeks before spinal surgery, were included in the HYADD® 3 material (1× 106 cells/ml). Two lumbar discs were operated in each animal. Control discs received a nucleotomy only. The animals were killed after 6 weeks and the lumbar spines recovered for histopathological study.

Intervertebral disc (IVD) damage due to degeneration, trauma or inflammation is the main cause for lower back pain leading to morbidity and loss of function of the spinal column. Until recently the state of the art treatment for degenerative disc disease (DDD) was arthrodesis. Developments in vertebral arthroplasty enable degenerated disc to be replaced with prosthetic IVD devices while maintaining motion at the affected part. The ability of the intervertebral device to support the in vivo loading environment is critical for the clinical success of such devices. However, such properties are depended on the location and structure of IVD, as the mechanical properties of IVD change locally [1]. The objective of this study was to evaluate the in vivo tissue compatibility of a novel composite, made with poly 2-hydroxyethyl methacrylate (pHEMA), poly ε-caprolactone (PCL) and poly ethylene terephthalate (PET) in an animal model. In vivo qualitative and quantitative results at 6 weeks post intraosseous implantation in rabbit femur revealed that this hydrogel, in contact with bone tissue, showed no tissue damage at the implant-bone interface. This novel composite disc prosthetic material is biocompatible as bone growth was observed into the implant and there was no evidence of toxicity to bone or inflammatory responses at the peri-implant tissue.

A review of the various coral bone graft substitutes currently available for experimental and biomedical applications and ongoing investigations of coral derived bone replacement materials is presented here. Natural and synthetic graft materials that have been studied in vitro and in vivo and used in different medical procedures in osseous tissue have focused mainly on freeze-dried bone, hydroxyapatite (HA), tricalcium phosphate (TCP) and coral. Coralline hydroxyapatite (CHA) is manufactured from marine coral, which has a natural trabecular structure similar to that of bone, by the hydrothermal conversion of the calcium carbonate skeleton of coral to hydroxyapatite, a calcium phosphate. While many studies have demonstrated promising biocompatible properties and osteogenic results, as a bone graft substitute and bone void filler, the use of CHA may be limited owing to its inherent mechanical weakness and reduced biodegradation. The benefits of CHA as bone graft are predominantly its safety, biocompatibility and osteoconductivity so that it can be used as a substitution biomaterial for bone in many indications clinically. CHA can also be used as an efficient carrier system for the local delivery of growth factors to enhance osteointegration and implant fixation into peri-implant osseous tissue.

Osteogenic growth factors are added to enhance osteointegration and osteogenesis of synthetic bone substitutes to improve clinical outcome. Reactions to particles of wear debris from implanted material could lead to bone resorption similar to resorption around a total joint prosthesis and also to inflammatory responses in distant organs. Porous hydroxyapatite (HA) scaffolds pretreated. with insulin like growth factor (IGF)-I (0.5 (LD) or 3.0 (HD) mug/implant) and IGF-II (0.5 mug/implant) were implanted in vivo in rabbit femur to enhance their bioactivity and bone bonding properties. Heart, kidney, liver, lung, lymph nodes and spleen were collected during systematic postmortem examination at 1, 3 and 5 weeks postimplantation for quantitative and qualitative analysis. Local wound healing of the periprosthetic bone and the responses in the distant internal organs were characterised using light microscopy and electron microscopy. All tissues from implanted groups except heart and kidneys exhibited an increase in the cellularity at week I and 3. In the lung, there was also evidence of lympho-proliferation and aggregation in the IGF groups and presence of exudates in the IGF-1 HD group. The hypertropby and hyperplasia appeared to be growth factor, dose and time dependent with IGF-1 LD <IGF-II <IGF-1 HD. At 5 weeks the tissues appeared as regenerated to the normal level with regression of the lymphocytic infiltration. We report for the first time, that local delivery of IGF-I and -II by intraosseous HA implant to enhance osteointegration produced no adverse persistent effects in important internal organs.

Synthetic hydroxyapatite, a bioactive calcium phosphate, is clinically used as a bone replacement bioceramic because of its similarity in composition to bone mineral, biocompatibility, and osteoconductive nature. The aim of this study was to evaluate the bioactivity of a novel synthetic porous hydroxyapatite (PHA) in vivo in rabbit and to investigate the enhancement of its bioactivity and osteointegration. In the investigation reported here, insulin-like growth factor-I (IGF-I) has been used to enhance the bioactivity of PHA. Cylindrical PHA implants with or without IGF-I were implanted bilaterally in rabbit femurs. Fluorochrome bone markers were administered at 7-day intervals. The implants with the attached bone were retrieved at postmortem, 1 and 3 weeks after implantation, for histological and histomorphometric analysis. Undecalcified sections stained with toluidine blue showed new bone formation. Mineralization of the new bone formed in the interface, surrounding trabecular bone, and within the pores of the implants was studied. Lamellar bone mineral apposition rate (MAR) was assessed and compared among treatment groups, sham, PHA alone, and PHA with IGF-I (500 ng/implant), by fluorochrome label incorporation using UVL microscopy. We report for the first time, that the supplementation of PHA implants with IGF-I significantly increased new bone formation and MAR (6.58 +/- 0.08 microm/day) compared with implantation of PHA alone (4.08 +/- 0.05 microm/day) or sham operation (3.11 +/- 0.12 microm/day). These results suggest that synthetic PHA might provide a delivery system for bioactive agents to accelerate bone healing in orthopedic procedures.

After 6 weeks implantation in the NZW rabbit, statistically significant enhanced bone bonding, measured as interfacial shear strength, has been demonstrated with Mg ion beam embedded HA coated TiAlV cylinders compared with an ordinary HA coating (p<0.05, n=7, student T test). The results are in keeping with previous studies of the effects of magnesium on bone cell activity.

Phase pure hydroxyapatite (HA) and a 0.8 wt % silicon substituted hydroxyapatite (SiHA) were prepared by aqueous precipitation methods. Both HA and SiHA were processed into granules 0.5–1.0 mm in diameter and sintered at 1200 °C for 2 h. The sintered granules underwent full structural characterization, prior to implantation into the femoral condyle of New Zealand White rabbits for a period of 23 days. The results show that both the HA and SiHA granules were well accepted by the host tissue, with no presence of any inflammatory cells. New bone formation was observed directly on the surfaces and in the spaces between both HA and SiHA granular implants. The quantitative histomorphometry results indicate that the percentage of bone ingrowth for SiHA (37.5%±5.9) was significantly greater than that for phase pure HA (22.0%±6.5), in addition the percentage of bone/implant coverage was significantly greater for SiHA (59.8%±7.3) compared to HA (47.1%±3.6). These findings indicate that the early in vivo bioactivity of hydroxyapatite was significantly improved with the incorporation of silicate ions into the HA structure, making SiHA an attractive alternative to conventional HA materials for use as bone substitute ceramics.

Mechanical strain, testosterone, and estrogen all stimulate proliferation of primary cultures of male rat long bone (LOB)-derived osteoblast-like cells as determined by [3H]thymidine incorporation. The maximum proliferative effect of a single period of mechanical strain (3400 microepsilon, 1 Hz, and 600 cycles) is additional to that of testosterone (10(-8) M) or estrogen (10(-8) M). The cells' proliferative response to strain is abolished both by concentrations of tamoxifen that cause proliferation (10(-8) M) and by those that have no effect (10(-6) M). Strain-related proliferation also is reduced by the estrogen antagonist ICI 182,780 (10(-8) M) but is unaffected by the androgen receptor antagonist hydroxyflutamide (10(-7) M). Tamoxifen, ICI 182,780, and the aromatase inhibitor 4-dihydroandrostenedione, at concentrations that have no effect on basal proliferation, significantly reduce the proliferative effect of the aromatizable androgen testosterone but not that of the nonaromatizable androgen 5alpha-dihydrotestosterone. Hydroxyflutamide, at a concentration that has no effect on basal proliferation (10(-7) M), eliminates the proliferative effect of 5alpha-dihydro-testosterone but had no significant effect on that caused by testosterone. Proliferation associated with strain is blocked by neutralizing antibody to insulin-like growth factor II (IGF-II) but not by antibody to IGF-I. Proliferation associated with testosterone is blocked by neutralizing antibody to IGF-I but is unaffected by antibody to IGF-II. These data suggest that in rat osteoblast-like cells from males, as from females, strain-related proliferation is mediated through the estrogen receptor (ER) in a manner that does not compete with estrogen but that can be blocked by ER modulators. Proliferation associated with testosterone appears to follow its aromatization to estrogen and is mediated through the ER, whereas proliferation associated with 5alpha-dihydrotestosterone is mediated by the androgen receptor. Strain-related proliferation in males, as in females, is mediated by IGF-II, whereas proliferation associated with estrogen and testosterone is mediated by IGF-I.

The estrogen receptor's role in bone cells' response to mechanical strain was investigated by studying the effect of the estrogen receptor modulators ICI 182, 780 and tamoxifen on the proliferation of primary cultures of rat long bone‐derived osteoblasts stimulated by the independent and combined effects of 17β‐estradiol, mechanical strain, and the mitogens basic fibroblast growth factor (bFGF), truncated insulin‐like growth factor (tIGF)‐I and tIGF‐II, and epidermal growth factor (EGF). 17β‐estradiol (10−10 M to 10−8 M) increased [3H]thymidine incorporation equally in cells from males and females, as did a single period of cyclical strain in the plastic strips onto which the cells had been seeded (peak strain 3,400 μϵ, 600 cycles, 1 Hz). At 10−8 M, neither ICI 182,780 nor tamoxifen had any effect on basal [3H]thymidine incorporation in these cells, but both compounds prevented their proliferative responses to 10−8 M 17β‐estradiol. Tamoxifen eliminated and ICI 182,780 substantially reduced the proliferation stimulated by strain. 17β‐estradiol partially rescued the strain‐related response from the effect of tamoxifen but not that of ICI 182,780. Both tamoxifen and ICI 182,780 reduced proliferation stimulated by 10−8 M EGF but had no effect on that by 10−7 M bFGF or tIGF‐I and tIGF‐II. That both ICI 182,780 and tamoxifen, which in other tissues act as estrogen antagonists, should reduce osteoblast proliferation stimulated by 17β‐estradiol and EGF, but not that by FGF or the IGFs, was expected since the mitogenic effects of estrogen and EGF involve the estrogen receptor, whereas those of FGF and the IGFs do not. That these compounds should prevent osteoblasts' proliferative response to strain suggests that strain also stimulates mitogenesis by a mechanism involving the estrogen receptor. If this is so, bones' reduced ability to maintain their structural strength after the menopause could be explained by less effective strain‐related (re)modeling when estrogen is absent and, among other changes, the estrogen receptor could be down‐regulated.

Gene transfer by intramuscular injection of plasmid DNA has potential application in gene therapy. We examined factors affecting the number of expressing fibers, in contrast to total expression, following injection of plasmid DNA. Barium chloride proved effective in inducing muscle necrosis and regeneration in mice, and this increased the number of fibers expressing a reporter gene. Coinjection of ion-channel modulators did not increase the number of positive fibers, but increasing dose and repeated administration of plasmid did. Importantly, the plasmid size (7-16 kb) did not affect the number of fibers expressing the transgene, in both normal and regenerating muscle.

Long‐term high‐level in vivo gene expression appears to depend on the promoter chosen to drive the gene of choice. In many cases the promoter appears to ‘switch off’ some time after in vivo gene transfer. We demonstrate that, following intramuscular injection of β‐galactosidase reporter plasmids, promoter ‘switch off’ is due to elimination of fibres expressing the transferred reporter gene by activation of a Th1 (cytotoxic) immune response. This finding, in the absence of stimulation of the immune system by viral vector proteins, has implications not only for gene transfer experiments but for the future of muscle‐directed gene therapy.