Longitudinal growth of mammalian bones: a possible role for membrane transporters in mediating chondrocyte hypertrophy

Long bone lengthening occurs at the growth plate (GP) by well-regulated chondrocyte proliferation, hypertrophy and terminal matrix deposition. GP chondrocyte (GPC) hypertrophy has been implicated to be the main determinant of bone growth rate; however the mechanism is poorly understood. The work of this thesis examined some of the cellular process that drives the chondrocyte swelling or hypertrophy particularly in a mammalian post natal GPs using living in situ GPC and fixed GP tissues. Confocal scanning microscopy (CLSM) was used to determine living in situ GPC volume and dimension changes in proliferative zone (PZ) through to hypertrophic zone (HZ) chondrocytes of different GPs of various bones. While PZ cells showed similar volumes and dimensions, HZ cells varied in different GPs, even within the same long bone but at opposite ends. However, the hypertrophic cell volume measured at a single post natal age (day 7) was independent of the corresponding bone length. This could reflect a complex interplay between local and systemic factors in different GPs, which occurs throughout the active phase of bone growth. Maintaining GPC morphology was critical in studying GPC hypertrophy using fixed tissues. This work highlighted a problem caused by conventional fixative solutions, which caused up to 44% hypertrophic GPC shrinkage following GP fixation. This artifact appeared to be associated with the hyperosmotic nature of the fixatives used and could be abolished by adjusting the fixative osmolarity close to physiological level (280 mOsm), or could be significantly reduced by bisecting bone tissues prior to tissue fixation. This thesis proposed roles for plasma membrane transporter(s) in mediating GPC hypertrophy. This hypothesis was tested by examining roles of sodium-hydrogen exchanger (NHE) and anion exchanger (AE) in GPC hypertrophy using an ex vivo bone growth inhibition model. Inhibition of bone growth by inhibitors of NHE (EIPA) and AE (DIDS) respectively was shown to be dose-dependent. The histology of bones demonstrated that the late HZ width was significantly reduced in GPs treated with EIPA or DIDS. Although in situ GPC volumes in the PZ and HZ were not notably different in DIDS-treated GP, the cell volumes in both zones were significantly reduced by EIPA treatment. Fluorescence immunohistochemistry revealed distinctive cellular localisations of NHE1 and AE2 in the PZ and early HZ. These results suggest a possible role of AE in mediating GPC volume increase in PZ chondrocytes and those in the early stages of cell hypertrophy, whereas NHE could possibly maintain intracellular pH of GPC throughout all GP zones. This thesis has characterized various changes in volume and dimensions of living in situ GPC from PZ through to HZ of different GPs of postnatal rats. This work emphasized the importance of fixative osmolarity in order to accurately preserve the normal volume/morphology of cells within tissues. Most importantly, this thesis confirmed a potential role of the plasma membrane transporters, AE and NHE in GPC hypertrophy of growing bones

Management of lower urinary tract obstructive disease using bladder tube cystotomy in a Saanen buck

This report describes a procedure of bladder tube cystotomy to relieve progressing bladder distension in a goat diagnosed to have obstructive urolithiasis. This one-year old Saanen buck was presented with a complaint of not passing urine and being in discomfort for almost three days. On physical examination, pulsation of the urethra in the perineal region could be felt and swelling and pain along the prepuce and perineum were indicated on deep palpation. Amputation of the urethral orifice and attempt to catheterise the urethra failed to relieve bladder distension; thus, bladder tube cystotomy procedure was performed. Following this procedure, all vital parameters returned to normal and normograde cystourethrogram was performed to radiographically assess the lower urinary tract of the goat. The outcome of this case supports that the bladder tube cystotomy technique provides an effective method to allow immediate relief of a distended bladder, allowing the urethra to heal as the urine outflow is diverted through the catheter and allows normograde cystourethrography to be performed

Scanning electron microscopic evaluation of natural coral (Porites sp.) post-implantation in sheep femur

The study was carried out with the aim to evaluate natural coral (Porites spp.) implanted in sheep femur using SEM. Twelve adult, male sheep divided into four groups (n=3) were used in this study. The large bone defect (2.5x0.5x05 cm) was created surgically under general anaesthesia on the left proximal femur. The defect area was implanted with coral and monitored for up to 12 weeks. Implants were harvested at 2, 4, 8 and 12 weeks intervals and subjected for scanning electron microscopy. The results showed that natural coral was found to be a biodegradable and osteo-conductive biomaterial, which acted as a scaffold for a direct osteoblastic apposition

Morphological features of natural cryptorchid sheep testes: a case study

The present study compares the morphological features of abdominal testes of natural bilateral cryptorchid adult sheep with that of scrotal testes in normal adult sheep. Specimens obtained from cryptorchid local sheep testes and epididymides were fixed and processed for light and scanning electron microscopy. Testes of normal sheep were fixed and processed in the same way and used for comparison. The retained testes that were detected in the pelvic cavity were small and flabby with indistinct superficial blood vessel ramification compared to the scrotal testes. Histologically, the seminiferous tubules of the retained testes were small, empty, lined with a single layer of degenerated cells resting on a thickened basement membrane surrounded with abundant interstitial tissue compared to the scrotal testis. The epididymis of the retained testes was empty and lined with high pseudostratified columnar epithelium with long streocillia, while the scrotal testis epididymis was distended with stored sperms and lined with low epithelial layer with indistinct streocillia. The connective tissue layers around the epididymis were very thick in retained testes compared to that of the scrotal testes. The outcome of this study demonstrates microscopic and macroscopic changes in cryptorchid testes. Further investigations are required regarding the ultrastructural changes in abdominal testis

Effect of preservation methods on the performance of bovine pericardium graft in a rat model

This study investigates the effect of preservation methods on the performance of bovine parietal pericardium grafts in a rat model. Mid-ventral full thickness abdominal wall defects of 3x2.5 cm in size were created in 90 male Sprague-Dawley rats (300-400 g), which were divided into three groups of 30 rats each. The abdominal defects of group one and two were repaired with lyophilized and glycerolized bovine pericardium grafts, while the defects of group three were repaired with expanded polytetrafluoroethylene (ePTFE) Mycro Mesh as a positive control. Another group of 30 rats underwent sham operation and was used for comparison as negative control. Each group of rats (n=30) was divided into five subgroups (n=6) and killed at 1, 3, 6, 9 and 18 weeks post-surgery for gross and morphological evaluations. The rats tolerated the surgical procedure well with a total mortality of 0.05%. No serious post-operative clinical complications or signs of rejection were encountered. Adhesions between the grafts and the underlying visceral organs observed in the study were mostly results of post-surgical complications. Glycerol preservation delayed degradation and replacement of the grafts, whereas lyophilization caused early resorption and replacement of the grafts. The glycerolized grafts were replaced with thick dense fibrous tissue, and the lyophilized grafts were replaced with thin loose fibrous tissue. The healing characteristic of the bovine pericardium grafts was similar to those of the sham-operated group, and quite different from those of the ePTFE Mycro Mesh. The outcome of the present study confirmed the superiority of glycerolized bovine pericardium grafts over its lyophilized counter part

Open-comminuted mid humeral fracture in a long-tailed macaque (Macaca fascicularis) – case report

A 2-year-old long-tailed macaque (Macaca fascicularis) was presented to the University Veterinary Hospital, Universiti Putra Malaysia for a traumatic injury to the left arm. Physical examination findings revealed a lacerated wound of 2 cm x 1 cm with distal humeral bone segment was protruding out from the muscles and skin. There were delayed pain sensation and withdrawal reflex of the left upper arm. Radiographic findings revealed discontinuity of the left humerus at the mid-shaft with a single fragment from the distal segment at the fracture site. The fracture was repaired with an open reduction and fixation technique with a combination of intramedullary pinning and cerclage wire. The patient regained motor function of the arm after six weeks of intramedullary pin implantation. Here we describe the severity of each grading system and the principles of open fracture management in macaques

Comparative evaluations of the processed bovine tunica vaginalis implant in a rat model

The aim of the present study was to evaluate the lyophilized and glycerolized bovine parietal tunica vaginalis for repair of a full-thickness abdominal wall defect and to compare their effectiveness with expanded polytetrafluoroethylene (ePTFE) Mycro Mesh (Gore-Tex® MYCRO MESH®; Gore and Designs, W. L. Gore & Associates, Flagstaff, AZ, USA) in a rat model. Fresh bovine parietal tunica vaginalis sacs collected from an abattoir were processed and preserved by freeze-drying and by using 99.5% glycerol. Full-thickness abdominal wall defects (3×2.5 cm) created surgically in 90 male Sprague-Dewaly rats (300–400 g) were repaired with the same size of lyophilized, glycerolized or ePTFE Mycro Mesh with 30 rats in each group. Six rats from each group were killed at post-implantation intervals of 1, 3, 6, 9 and 18 weeks for macroscopic, microscopic and tensiometric evaluations. All rats survived the procedure, except for one rat in the ePTFE Mycro Mesh group. Implants of bovine origin were gradually resorbed and replaced with recipient fibrous tissue, whereas the mesh implant was encapsulated with fibrous tissue and remained without any marked changes throughout the study period. Adhesions between the implant and underlying visceral organs were encountered in 10, 6.6 and 3.3% of rats implanted with lyophilized, glycerolized or ePTFE Mycro Mesh, respectively. Foreign body giant cells and calcification were demonstrated in fibrous capsule and mesh matrix, respectively, in rats implanted with ePTFE Mycro Mesh. Neither of these characteristics were observed in rats implanted with processed bovine tunica vaginalis. Macrophages engorged with lipofuchsin pigments were observed in the recipient tissue that replaced the implants of bovine origin at 3–18 weeks post-implantation. There were no significant (P > 0.05) differences in total mean values of healing tensile strength, load at break and Young’s modulus of elasticity between the three implant groups. The results of the present study encourage further investigation into the use of processed bovine parietal tunica vaginalis in clinical practice

Evaluation of rat soft tissue response to implantation of glycerolized bovine tunica vaginalis

The aim of this study was to evaluate glycerolized bovine parietal tunica vaginalis implant in rat model. Pieces of 3_2.5 cm bovine parietal tunica vaginalis preserved in 99.5% glycerol and stored at 4 degrees C were used to repair 3_2.5 cm full thickness abdominal wall defects created in a group of 30 male Sprague Dawley rats (300-400 g). Another group of 30 rats were underwent sham operation and used for comparison. Each group was divided into five subgroups (n=6) and sacrificed at post-surgical intervals of 1, 3, 6, 9 and 18 weeks for macroscopical, histological and mechanical evaluation. Loose adhesions were observed between the implanted graft and underlying visceral organs in 6.6% of the treated group. Histologically the graft was biocompatible and gradually replaced by the recipient fibers tissue. The graft healing tensile strength increased with time in both groups and no significant different (P>0.05) was observed between the overall means of healing tensile strength of the two groups. The outcome of this study revealed that glycerolized bovine tunica vaginalis is biocompatible surgical patch that can be used for reconstruction of soft tissue defects. However, further investigation is required regarding the glycerol preservation efficiency