ABSTRACT
Testosterone and alendronate have been identified as two bone healing compounds which, when combined, synergistically stimulate bone regeneration. This study describes the development of a novel ultrasonic spray coating for sustained release of ancillary amounts of testosterone and alendronate encapsulated in PLGA 5004A as a carrier. Due to the low amounts of testosterone and alendronate used, sensitive in vitro assays were developed to determine in vitro release. The ultrasonic spray coating technology was optimized for coating titanium screws and pericardial collagen membranes, with the aim to improve osseo-integration and (guided) bone regeneration, respectively, without interfering with their primary mode of action. In vitro release analysis of collagen membranes and screws showed up to 21 days sustained release of the compounds without a burst release. Subsequent preclinical studies in rat and rabbit models indicated that testosterone and alendronate coated membranes and screws significantly improved bone regeneration in vivo. Coated membranes significantly improved the formation of new bone in a critical size calvarial defect model in rats (by 160% compared to controls). Coated screws implanted in rabbit femoral condyles significantly improved bone implant contact (69% vs 54% in controls), bone mineral density (121%) and bone volume (119%) up to 1.3 mm from the implant. Based on the results obtained, we suggest that implants or membranes enabled with local sustained delivery of ancillary amounts of testosterone and alendronate can be a promising system to stimulate local bone regeneration resulting in improved osseo-integration of implants and improved healing of bone defects and fractures.
Subject(s)
Alendronate/pharmacology , Bone Regeneration/drug effects , Osseointegration/drug effects , Testosterone/pharmacology , Animals , Bone Density/drug effects , Bone Density Conservation Agents/chemistry , Bone Density Conservation Agents/pharmacology , Bone Regeneration/physiology , Coated Materials, Biocompatible/chemistry , Coated Materials, Biocompatible/pharmacology , Delayed-Action Preparations/pharmacology , Disease Models, Animal , Femur/growth & development , Femur/surgery , Humans , Male , Osseointegration/physiology , Polylactic Acid-Polyglycolic Acid Copolymer/chemistry , Polylactic Acid-Polyglycolic Acid Copolymer/pharmacology , Prostheses and Implants , Rabbits , Rats , Titanium/chemistry , Titanium/therapeutic useABSTRACT
Liver receptor homologue-1 (LRH-1) is an orphan nuclear receptor that has been implicated in steroid hormone biosynthesis and fertility. Herein we describe a transgenic inducible short hairpin (sh) RNA mouse model that was used to study the effect of transient LRH-1 knockdown in vivo. Induction of expression of the shRNA directed against LRH-1 for 2-6 weeks resulted in 80% knockdown of LRH-1 protein in the ovary and complete infertility. Gonadotropin hyperstimulation could not rescue the observed defects in ovulation and corpus luteum formation in LRH-1-knockdown mice. The infertility phenotype was fully reversible because LRH-1-knockdown females became pregnant and delivered normal size litters and healthy pups after cessation of LRH-1 shRNA expression. Timed ovarian microarray analysis showed that, in line with the observed decrease in plasma progesterone levels, key steroid biosynthesis genes, namely Star, Cyp11a1, Hsd3b and Scarb1, were downregulated in LRH-1-knockdown ovaries. In contrast with what has been described previously, no clear effect was observed on oestrogenic activity in LRH-1-knockdown mice. Only Sult1e1 and, surprisingly, Hsd17b7 expression was modulated with potentially opposite effects on oestradiol bioavailability. In conclusion, the fully reversible infertility phenotype of LRH-1-knockdown mice shows the feasibility of an LRH-1 antagonist as new contraceptive therapy with a mechanism of action that most prominently affects cholesterol availability and progesterone production.
Subject(s)
Fertility , Gene Knockdown Techniques , Infertility, Female/metabolism , Ovary/metabolism , Receptors, Cytoplasmic and Nuclear/deficiency , Animals , Cells, Cultured , Cholesterol/metabolism , Estradiol/metabolism , Female , Gene Expression Regulation, Enzymologic , Genotype , Infertility, Female/genetics , Infertility, Female/physiopathology , Mice , Mice, Transgenic , Ovary/physiopathology , Phenotype , Pregnancy , Progesterone/metabolism , RNA Interference , RNA, Small Interfering/genetics , Receptors, Cytoplasmic and Nuclear/genetics , Time FactorsABSTRACT
CXCR7 is a G-protein coupled receptor that was recently deorphanized and shown to have SDF1 and I-TAC as high affinity ligands. Here we describe the characterization of CXCR7-deficient mice that were generated to further investigate the function of this receptor in vivo. Expression analysis using a LacZ reporter knockin revealed that postnatally Cxcr7 was specifically expressed in cardiomyocytes, vascular endothelial cells of the lung and heart, the cerebral cortex and in osteocytes of the bone. Adult tissues revealed high expression in cardiomyocytes and osteocytes. The observation that 70% of the Cxcr7-/- mice died in the first week after birth coincides with expression of Cxcr7 in vascular endothelial cells and in cardiomyocytes. An important role of CXCR7 in the cardiovascular system was further supported by the observation that hearts of the Cxcr7-/- mice were enlarged, showed myocardial degeneration and fibrosis of postnatal origin, and hyperplasia of embryonic origin. Despite high expression in osteocytes no apparent bone phenotype was observed, neither in combination with ovariectomy nor orchidectomy. Thus as CXCR7 does not seem to play an important role in bone our data indicate an important function of CXCR7 in the cardiovascular system during multiple steps of development.
