Research On Gene Therapy Of Postmenopausal Osteoporosis With Microencapsulated Genetically Modified Cells Expressing Human Calcitonin

Background: Osteoporosis is a disease of reduced skeletal mass and microarchitectural deterioration of the skeleton, resulting in an increased risk of fractures. Postmenopausal osteoporosis is the most common type of the disease. Fractures associated with osteoporosis and osteopenia are associated with significant increases in morbidity and mortality. Fractures resulting from low bone mass also have a significant impact on life quality of patients and social health care cost. The loss of ovarian hormone production in menopause is the major risk factor for osteoporosis, causing increased skeletal resorption and relatively decreased bone formation, continuing until the end of life. The increased bone remodeling, due to excessive osteoclast activity and an insufficiency of bone formation, results in deep resorption cavities, trabecular plate perforation, wide separation and disconnection of trabecular, and enlargement and coalescence of subendocortical spaces. Due to bone high-turnover rate after the menopause, antiresorptive drugs are reasonable choice for physicians. Several alternative treatments are available,including established drug therapies such as hormone replacement therapy,bisphosphonates, selective estrogen receptor modulators as well as calcitonin. Calcitonin (CT) is a peptide hormone produced by the parafollicular cells of the thyroid gland in mammals, which directly inhibits osteoclastic bone resorption. In spite of the novel therapeutic effect of calcitonin on bone diseases, the exact role of calcitonin in the pathogenesis of postmenopausal osteoporosis remains controversial. While , the use of these therapeutic proteins in the treatment of postmenopausal osteoporosis is limited by the need for repeated protein administration, costly production methods and antigenicity.Objective: The purpose of the first part of this study was to investigate the changes of morphology and calcitonin secretory reserve of thyroid C cells in ovariectomized rats, in aiming to study the role of calcitonin in the pathogenesis of postmenopausal osteoporosis. Meanwhile,Mouse myoblasts strain which steadily expressed human calcitonin were established using the cationic liposome-based gene delivery technique. The hCT cDNA transfected myoblasts were encapsulated in non-antigenic biocompatible alginate-polylysine-alginate microcapsules. The effects of recombinant human calcitonin secreted from the cells on osteoblasts and osteoclasts were investigated in vitro. Then, Polymer microcapsules loaded with hCT-secreting myoblasts were implanted in the peritoneum of the ovariectomized rats to examine the therapeutic feasibility of the protocol.Method: Firstly, sixteen 6-month-old, female SD rats were divided into two groups. One was non-ovariectomized group (Sham) and the other was ovariectomized group (OVX). Twelve weeks after surgery, the alterations of thyroid C cell secretory reserve function in two groups were assessed by the calcium infusion test. A peroxidase–antiperoxidase method was applied for localization of calcitonin (CT) in the C cells. Morphometric changes in their volume,relative volume density and numerical density were evaluated in comparison with sham-operated control rats using a stereological method. Then, Mouse myoblasts were transfected with the cDNA for human calcitonin using the cationic liposome-based gene delivery technique and clones secreting high levels of human calcitonin were isolated. The expression and secretion of human calcitonin by myoblast cells was confirmed by RT-PCR,ELISA,Western blot analysis and immunohistochemical analyses. Upon enclosure in alginate-polylysine-alginate microcapsules, which are biocompatible membranes that permit exit of therapeutic protein but not entry of immune mediators, the encapsulated myoblasts were cultured in vitro. Primary osteoblasts and osteoclasts were subjected to recombinant human calcitonin secreted from the cells at different concentrations. The effects of recombinant peptide on proliferation and function of primary osteoblasts and osteoclasts were studied. Then, Polymer microcapsules loaded with hCT-secreting myoblasts were implanted in the peritoneum of the ovariectomized rats. The concentration of human calcitonin was measured by ELISA. Bone mineral density of lumbar was determined by DEXA. Biomechanical property of lumbar and femur were also examined. Some biochemical markers of bone metabolism as serum calcium,serum inorganic phosphorus,serum osteocalcin (BGP) and unrinary pyridinoline (PYD) were examined. Bone histomorphometry,Light microscope and Scanning Electronic Microscopy were used to investigated skeletal changes in cancellous bone in section of lumbar and tibia.Results:⑴Ovariectomy led to trabecular volume and number, wide separation and disconnection of trabecular in section of lumbar. The fact indicated the animal model of osteoporosis had been duplicated successfully. In ovariectomized rats, the three parameters as basal CT, the peak CT value and the CT increase range were significantly lower than those in normal controls. The number of thyroid C cells of the ovariectomized rats was markedly increased.⑵Reverse transcriptive-polymerase chain reaction (RT-PCR) and enzyme immunoassay confirmed that transgenes were expressed in the myoblast cells. Meanwhile, Western blot and immunohistochemical analyses detected significant increases in human calcitonin levels in the transfected myoblasts.⑶The encapsulated recombinant myoblasts continued to secrete human calcitonin in vivo and in vitro.⑷The recombinant protein showed marked increases in osteoblast proliferation, alkaline phosphates activity and formation of mineral nodules in dosage-dependant manner.⑸The recombinant human calcitonin decreased the number of osteoclasts and caused a dose-dependent decrease in mature osteoclasts activity as measured by the area of bone resorptive pits.⑹The bone turnover rate in ovariectomized rats had decreased after implantation. In comparison with the osteoporotic rats, those implanted with encapsulated recombinant myoblasts showed less bone loss; the reduction in bone loss was obvious in vertebral body, but this was hardly seen in the proximal tibial metaphases. Histological findings of lumbar vertebra revealed the improvement of bone architect of the axial bone. Conclusion:⑴The deficiency of synthesis and release of calcitonin in the thyroid C cells may account partially for the bone loss in ovariectomized rats.⑵The encapsulated recombinant myoblasts can serve as an efficient and stable delivery vehicle producing human calcitonin.⑶The recombinant protein has positive effects on proliferation, ALP activity and mineralizing nodules formation in osteoblasts. It decreases the number of osteoclasts and inhibited their bone resorptive function.⑷The recombinant human calcitonin secreted from the encapsulated myoblasts can reverse the bone loss in axial bone due to ovariectomy.