Pre-grafting histological studies of skin grafts cryopreserved in α helix antarctic yeast oriented antifreeze peptide (Afp1m).

A number of living creatures in the Antarctic region have developed characteristic adaptation of cold weather by producing antifreeze proteins (AFP). Antifreeze peptide (Afp1m) fragment have been designed in the sequence of strings from native proteins. The objectives of this study were to assess the properties of Afp1m to cryopreserve skin graft at the temperature of -10 °C and -20 °C and to assess sub-zero injuries in Afp1m cryopreserved skin graft using light microscopic techniques. In the present study, a process was developed to cryopreserve Sprague-Dawley (SD) rat skin grafts with antifreeze peptide, Afp1m, α-helix peptide fragment derived from Glaciozyma antractica yeast. Its viability assessed by different microscopic techniques. This study also described the damages caused by subzero temperatures (-10 and -20 °C) on tissue cryopreserved in different concentrations of Afp1m (0.5, 1, 2, 5 and 10 mg/mL) for 72 h. Histological scores of epidermis, dermis and hypodermis of cryopreserved skin grafts showed highly significant difference (p < 0.01) among the different concentrations at -10 and -20 °C. In conclusion, the integrity of cryopreserved skin grafts with lower concentrations of Afp1m (0.5, 1 and 2 mg/mL) or at -20 °C was not maintained. The present study attested that Afp1m is a good cryoprotective agent for the cryopreservation of skin graft. Higher Afp1m concentrations (5 and 10 mg/mL) at -10 °C found to be suitable for the future in vivo study using (SD) rat skin grafts.

Histological and mechanical evaluation of antifreeze peptide (Afp1m) cryopreserved skin grafts post transplantation in a rat model.

The objective of this study was to evaluate the use of Afp1m as a cryopreservative agent for skin by examining the transplanted skin histological architecture and mechanical properties following subzero cryopreservation. Thirty four (34) rats with an average weight of 208 ±â€¯31 g (mean ±â€¯SD), were used. Twenty four (n = 24) rats were equally divided into four groups: (i) immediate non-cryopreserved skin autografts (onto same site), (ii) immediate non-cryopreserved skin autografts (onto different sites), (iii) skin autografts cryopreserved with glycerol for 72 h and (iv) skin autografts cryopreserved with Afp1m for 72 h at -4 °C. Rounded shaped full-thickness 1.5-2.5 cm in diameter skin was excised from backs of rats for the autograft transplantation. Non-cryopreserved or cryopreserved auto skin graft were positioned onto the wound defects and stitched. Non-transplanted cryopreserved and non-cryopreserved skin strips from other ten rats (n = 10) were allowed for comparative biomechanical test. All skin grafts were subjected to histological and mechanical examinations at the end of day 21. Histological results revealed that tissue architecture especially the epidermal integrity and dermal-epidermal junction of the Afp1m cryopreserved skin grafts exhibited better histological appearance, good preservation of tissue architecture and structural integrity than glycerolized skin. However, there was no significant difference among these groups in other histological criteria. There were no significant differences among the 4 groups in skin graft mechanical properties namely maximum load. In conclusion, Afp1m were found to be able to preserve the microstructure as well as the viability and function of the skin destined for skin transplantation when was kept at -4 °C for 72 h.

Trends of reactive hyperaemia responses to repetitive loading on skin tissue of rats - Implications for pressure ulcer prevention.

Tissue recovery is important in preventing tissue deterioration, which is induced by pressure and may lead to pressure ulcers (PU). Reactive hyperaemia (RH) is an indicator used to identify people at risk of PU. In this study, the effect of different recovery times on RH trend is investigated during repetitive loading. Twenty-one male Sprague-Dawley rats (seven per group), with body weight of 385-485 g, were categorised into three groups and subjected to different recovery times with three repetitive loading cycles. The first, second, and third groups were subjected to short (3 min), moderate (10 min), and prolonged (40 min) recovery, respectively, while fixed loading time and pressure (10 min and 50 mmHg, respectively). Peak hyperaemia was measured in the three cycles to determine trends associated with different recovery times. Three RH trends (increasing, decreasing, and inconsistent) were observed. As the recovery time is increased (3 min vs. 10 min vs. 40 min), the number of samples with increasing RH trend decreases (57% vs. 29% vs. 14%) and the number of samples with inconsistent RH trend increases (29% vs. 57% vs. 72%). All groups consists of one sample with decreasing RH trend (14%). Results confirm that different recovery times affect the RH trend during repetitive loading. The RH trend may be used to determine the sufficient recovery time of an individual to avoid PU development.

The use of rats and mice as animal models in ex vivo bone growth and development studies.

In vivo animal experimentation has been one of the cornerstones of biological and biomedical research, particularly in the field of clinical medicine and pharmaceuticals. The conventional in vivo model system is invariably associated with high production costs and strict ethical considerations. These limitations led to the evolution of an ex vivo model system which partially or completely surmounted some of the constraints faced in an in vivo model system. The ex vivo rodent bone culture system has been used to elucidate the understanding of skeletal physiology and pathophysiology for more than 90 years. This review attempts to provide a brief summary of the historical evolution of the rodent bone culture system with emphasis on the strengths and limitations of the model. It encompasses the frequency of use of rats and mice for ex vivo bone studies, nutritional requirements in ex vivo bone growth and emerging developments and technologies. This compilation of information could assist researchers in the field of regenerative medicine and bone tissue engineering towards a better understanding of skeletal growth and development for application in general clinical medicine.Cite this article: A. A. Abubakar, M. M. Noordin, T. I. Azmi, U. Kaka, M. Y. Loqman. The use of rats and mice as animal models in ex vivo bone growth and development studies. Bone Joint Res 2016;5:610-618. DOI: 10.1302/2046-3758.512.BJR-2016-0102.R2.

A cell shrinkage artefact in growth plate chondrocytes with common fixative solutions: importance of fixative osmolarity for maintaining morphology.

The remarkable increase in chondrocyte volume is a major determinant in the longitudinal growth of mammalian bones. To permit a detailed morphological study of hypertrophic chondrocytes using standard histological techniques, the preservation of normal chondrocyte morphology is essential. We noticed that during fixation of growth plates with conventional fixative solutions, there was a marked morphological (shrinkage) artifact, and we postulated that this arose from the hyper-osmotic nature of these solutions. To test this, we fixed proximal tibia growth plates of 7-day-old rat bones in either (a) paraformaldehyde (PFA; 4%), (b) glutaraldehyde (GA; 2%) with PFA (2%) with ruthenium hexamine trichloride (RHT; 0.7%), (c) GA (2%) with RHT (0.7%), or (d) GA (1.3%) with RHT (0.5%) and osmolarity adjusted to a 'physiological' level of approximately 280mOsm. Using conventional histological methods, confocal microscopy, and image analysis on fluorescently-labelled fixed and living chondrocytes, we then quantified the extent of cell shrinkage and volume change. Our data showed that the high osmolarity of conventional fixatives caused a shrinkage artefact to chondrocytes. This was particularly evident when whole bones were fixed, but could be markedly reduced if bones were sagittally bisected prior to fixation. The shrinkage artefact could be avoided by adjusting the osmolarity of the fixatives to the osmotic pressure of normal extracellular fluids ( approximately 280mOsm). These results emphasize the importance of fixative osmolarity, in order to accurately preserve the normal volume/morphology of cells within tissues.

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 3 x 2.5 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.

Bone graft substitute using hydroxyapatite scaffold seeded with tissue engineered autologous osteoprogenitor cells in spinal fusion: early result in a sheep model.

Spinal fusion using autologous bone graft is performed in an increasing rate for many spinal disorders. However, graft harvesting procedure is associated with prolonged operation time and potential donor site morbidity. We produced an engineered 'bone graft' substitute by using porous hydroxyapatite (HA) scaffold seeded with autologous bone marrow osteoprogenitor cells (OPCs) and fibrin. This obviates bone graft harvesting, thus eliminates donor site morbidity and shortens the operation time. The aim of this study is to evaluate Hydroxyapatite (HA) ceramics as scaffold for autologous tissue engineered bone construct for spinal fusion in a sheep model. The sheep's marrow was aspirated from iliac crest. The bone marrow mesenchymal stem cells (BMMSCs) were cultured for several passages in the presence of growth and differentiation factors to increase the number of OPCs. After the cultures reached confluence, they were trypsinized and seeded on Hydroxyapatite scaffold (HA). Approximately 5 million cells were generated after 3 weeks of culture. Microscopically, very tight Colony Forming Units (CFU-Fs) were seen on monolayer culture. The Von Kossa and Alizarin Red staining of monolayer culture showed positive mineralization areas; indicating the presence of OPCs. Sheep underwent a posterolateral spinal fusion in which scaffolds with or without OPCs seeded were implanted on both sides of the lumbar spine (L1-L2). Intended fusion segments were immobilized using wires. At the end of third month, the fusion constructs were harvested for histological examination. Fibrous tissue infiltration found in the inter-connecting pores of plain HA ceramics indicates inefficient new bone regeneration. New bone was found surrounding the HA ceramics seeded with autologous cells. The new bone is probably formed by the sheep BMMSCs that were initially encapsulating HA while it remained intact. The new bone is naturally fused with the vertebrae. In conclusion, the incorporation of autologous bone marrow cells improved the effectiveness of HA ceramics as 'bone graft' substitute for spinal fusion.

Effect of freeze-drying and gamma irradiation on biomechanical properties of bovine pericardium.

Freeze-drying and gamma irradiation are the techniques widely use in tissue banking for preservation and sterilization of tissue grafts respectively. However, the effect of these techniques on biomechanical properties of bovine pericardium is poorly known. A total of 300 strips of bovine pericardium each measured 4 cm x 1 cm were used in this study to evaluate the effect of freeze-drying on biomechanical properties of fresh bovine pericardium and the effect of gamma irradiation on biomechanical properties of freeze-dried bovine pericardium. The strips were divided into three equal groups, which consist of 100 strips each group. The three groups were fresh bovine pericardium, freeze-dried bovine pericardium and irradiated freeze-dried bovine pericardium. The biomechanical properties of the pericardial strips were measured by a computer controlled instron tensiometer while the strips thickness was measured by Mitutoyo thickness gauge. The results of the study revealed that freeze-drying has no significant (p > 0.05) effect on the tensile strength, Young's modulus (stiffness) and elongation rate of fresh bovine pericardium. Irradiation with 25 kGy gamma rays caused significant decreased in the tensile strength, Young's modulus and elongation rate of the freeze-dried pericardium. However, gamma irradiation has no significant effect on the thickness of freeze-dried bovine pericardium, while freeze-drying caused significant decreased in the thickness of the fresh bovine pericardium. The outcome of this study demonstrated that freeze-drying has no significant effect on the biomechanical properties of fresh bovine pericardium, and gamma irradiation caused significant effect on the biomechanical properties of freeze-dried bovine pericardium.

The use of freeze-dry bovine pericardium (FDBP) in diaphragmatic herniorrhaphy in dogs.

A study was conducted to investigate the effectiveness of freeze-dried bovine pericardium (FDBP) as a biomaterial in diaphragmatic herniorrhapy in dogs. Eight adult dogs were randomly selected and divided into two equal groups. In FDBP group, a diaphragmatic defect was induced and repaired with an identical size of FDBP. In the control group, a diaphragmatic wall was incised at three-side border creating a flap and sutured. Grossly, only mild intrathoracic adhesion was observed for most of the animals, and no herniation occured. Microscopically, the biomaterial incorporated into the host's tissue by ingrowth of young muscle fiber and massive new blood vessel formation in between the fibrous tissue.

Glycerol preserved bovine pericardium for abdominal wall reconstruction: experimental study in rat model.

The aim of this study was to evaluate bovine pericardium surgical patch in rat model. Bovine pericardial sacs collected from local abattoir were cleaned, disinfected and cut into pieces of 3 by 2.5cm and preserved in 99.5% glycerol. Full thickness abdominal wall defects of 3 by 2.5 cm were created in 30 adult male Sprague Dawley rats and repaired with glycerol preserved pieces. The rats were serially sacrificed in a group of six rats at 1,3,6,9 and 18 weeks post-surgical intervals for morphological and tensometeric study. Macroscopically, no mortality or postoperative surgical complications was encountered except slight adhesions between implanted grafts and some visceral organs in 10% of the rats. Microscopically no calcification or foreign body giant cell formation was found in the explanted grafts. The implanted grafts were replaced gradually with recipient tissue, which made mainly of dense collagenous bundles. The healing strength between the implanted grafts and the recipient abdominal wall was gradually increased with time. The results of this study showed that glycerol preserved bovine pericardium act as scaffold for transformation into living tissue without clinical complications such as that associated with prostheses.

Microscopic evaluation of the natural coral (Porites spp.) post-implantation in sheep femur.

The study was carried out with the aim to evaluate natural coral (Porites spp.) implanted in sheep femur microscopically. Twelve adult, male sheep were used in this study. The defect area was implanted with coral and monitored for up to 12 weeks. The sheep were euthanased at 2,4,8, and 12 weeks post-implantation. Microscopically, natural coral implanted into bone tissue have shown gradual resorption and progressively replaced by new bone. At 12 weeks post-implantation, the implanted site was almost completely surrounded by mature bone. 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.

Gross, radiology and ultrasonographic evaluation of coral post-implantation in sheep femur.

The study was carried out to evaluate macroscopically the ability of coral to repair a large size bone defect. A total 12 adult, male sheep were used in the study. The large bone defect (2.5cm x 0.5cm x 0.5cm) was created surgically on the left proximal femur and replaced by a block of coral (Porites sp.). Radiographs were obtained immediately after surgery and at 2, 4, 8 and 12 weeks post-implantation. Ultrasonographic examinations were carried out every 2 weeks after implantation up to 12 weeks using ultrasound machine (TOSHIBA Capasee II) connected with 7MHz frequency transducer. The sheep were euthanased at 2, 4, 8, and 12 weeks post-implantation and the bone examined grossly. Both ultrasonographs and radiographs taken at 8 and 12 weeks showed that the implants had been resorbed and left the space that much reduced in size. There was no sign of implant rejection observed in all animals. The results showed that processed coral has potential to become bone substitute for reconstructive bone surgery.