The reduced stability of hydroxyapatite (HA)-coated implants in osteopenic conditions is considered to be a major problem. We therefore developed a model of a boosted cementless implantation in osteopenic rats. Twelve-week-old rats were either ovariectomised (OVX) or sham-operated (SO), and after 24 weeks plain or HA-coated implants were inserted. They were treated with either a prostaglandin EP4 receptor agonist (ONO-4819) or saline for one month. The EP4 agonist considerably improved the osteoporosis in the OVX group. Ultrastructural analysis and mechanical testing showed an improvement in the implant-bone attachment in the HA-coated implants, which was further enhanced by the EP4 agonist. Although the stability of the HA-coated implants in the saline-treated OVX rats was less than in the SO normal rats, the administration of the EP4 agonist significantly compensated for this shortage. Our results showed that the osteogenic effect of the EP4 agonist augmented the osteoconductivity of HA and significantly improved the stability of the implant-bone attachment in the osteoporotic rat model

1. Tetracycline labelling methods have been used to measure the rate of growth in length and the rate of growth in width of the tibia of the normal rat. 2. The main limitations of the tetracycline methods are that in very young animals the bands of labelled bone are indistinct and remodelling occurs quickly; in animals nearing maturity, the growth in width is very slow and periods of at least fourteen days are required to give reliable results. 3. The tetracycline labelling methods can be used also to determine changes in the basic processes of bone formation and bone resorption

Rat patellae were preincubated with culture medium M199 for one hour and then with either fresh culture medium or Ringer's solution, Ringer lactate, Ringer glucose, normal saline or Betadine for another hour. The rate of proteoglycan synthesis in the articular cartilage was then measured by uptake of 35SO4 for the next 16 hours. Cartilage metabolism was inhibited by all of the solutions even after a recovery time of 16 hours. The inhibition was by 5% for Ringer's solution, 10% for Ringer glucose (p < 0.01), 20% for saline and Ringer lactate (p < 0.001) and 55% for Betadine (p < 0.001). Ringer's solution is therefore the best choice for joint irrigation during arthroscopy or other procedures

The presence of the connective tissue components fibronectin and the different types of collagen was demonstrated by histological and immunohistological methods in the granulation and scar tissue of a healing injury in rat muscle. The effects of physical activity on granulation tissue production, scar formation and muscle regeneration at various stages of healing were studied. It was shown that immobilisation after injury accelerates granulation tissue production, but if continued too long, leads to contraction of the scar and to poor structural organisation of the components of regenerating muscle and scar tissue. However, a certain period of immobilisation, about five days for rat muscle, is required to allow newly-formed granulation tissue to cover the injured area and to have sufficient tensile strength to withstand subsequent mobilisation. This mobilisation, at the correct interval, seems essential for the quicker resorption of scar tissue and the better structural organisation of the muscle

The effects of extracorporeal shock waves (ESWT) on tendon healing were assessed by observing histological and biomechanical parameters in a rat model of injury to the tendo Achillis. The injury was created by inserting an 18-G needle through tendo Achillis in 48 adult Wistar albino rats. The animals were divided into three groups. The first group received radiation only after the operation. The second received no shock waves and the third had 500 15 KV shocks on the second post-operative day. All the rats were killed on the 21st day after surgery. Histopathological analysis showed an increase in the number of capillaries and less formation of adhesions in the study group compared with the control group (p = 0.03). A significantly greater force was required to rupture the tendon in the study group (p = 0.028). Our findings suggest a basis for clinical trials using ESWT

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.

Intermittent treatment with parathyroid hormone (PTH) has an anabolic effect on both intact cancellous and cortical bone. Very little is known about the effect of the administration of PTH on the healing of fractures or the incorporation of orthopaedic implants. We have investigated the spontaneous ingrowth of callus and the formation of bone in a titanium chamber implanted at the medioproximal aspect of the tibial metaphysis of the rat. Four groups of ten male rats weighing approximately 350 g were injected with human PTH (1-34) in a dosage of 0, 15, 60 or 240 μg/kg/day, respectively, for 42 days from the day of implantation of the chamber. During the observation period the chamber became only partly filled with callus and bone and no difference in ingrowth distance into the chamber was found between the groups. The cancellous density was increased by 90%, 132% and 173% in the groups given PTH in a dosage of 15, 60 or 240 μg/kg/day, respectively. There was a linear correlation between bone density and the log PTH doses (r. 2. = 0.6). Our findings suggest that treatment with PTH may have a potential for enhancement of the incorporation of orthopaedic implants as well as a beneficial effect on the healing of fractures when it is given in low dosages

Objectives

Insufficient protein ingestion may affect muscle and bone mass, increasing the risk of osteoporotic fractures in the elderly, and especially in postmenopausal women. We evaluated how a low-protein diet affects bone parameters under gonadal hormone deficiency and the improvement led by hormone replacement therapy (HRT) with 17β-oestradiol.

We studied the sensory afferent properties of normal, immobilised and inflamed rat knees by recording the activity of the medial articular nerve (MAN). When the knee was inflamed by kaolin-carrageenan or immobilised for six weeks, MAN activity significantly increased during rest and continuous passive motion (CPM). The maximal discharge rate tended to increase depending on the angular velocity of the CPM. When the knees were then rested for one hour before again starting CPM, activity was further increased at the initial CPM cycle, the ‘post-rest effect’. Analysis of the conduction velocity showed that 94% and 66% of spike units on the recorded discharge of the immobilised and inflamed knees, respectively, belonged to fine nerve fibres. Our findings show that the sensory receptors in the knee are sensitised in a similar manner by immobilisation and by inflammation, suggesting a relationship to pain. The post-rest effect may be related to a characteristic symptom of osteoarthritis called ‘starting pain’

When large daily doses of vitamin D were administered to rats endochondral growth was inhibited and bone resorption occurred; later in the process uncalcified matrix (osteoid) like that seen in rickets formed on trabecular margins. When vitamin D was given only for a short period and then discontinued, little resorption of bone was seen during the withdrawal period and wide seams of osteoid material appeared which eventually calcified in an irregular manner. When normal endochondral growth was resumed a wide transverse band of dense bone with enclosed cartilaginous cores was left in the marrow cavity. If, after a few days, a second large dose of the vitamin was given resorption again occurred and calcification of osteoid material was accelerated, the first microscopic sign being a dense, wide, granular, deeply staining line at the junction of the bone and new osteoid. After a second withdrawal period a second layer of osteoid formed; eventually another transverse band appeared in the metaphysis. If this hypervitaminosis D cycle (+4 -12) was continued rats continued to form new bone with relatively little remodelling, so that after three such cycles bones became dense and hard. Histological study showed that little marrow cavity remained in either skull, vertebrae or epiphyses and a dense mass of bone enclosing cartilage cores filled the metaphysial part of the long bones. In addition, ankylosis ofteeth, calcification of spinal ligaments and widespread metastatic calcification were present. When hypervitaminosis D cycles (+1 -12, +1 -21) were adjusted to produce minimal resorptive changes a wide range of bone change was observed. This varied from uniform dense metaphysial bone containing abnormal cartilage matrix arranged in longitudinal striations, dense transverse bands parallel to the epiphysial cartilage, to remnants of dense trabeculae extending into the marrow cavity. Bone changes in osteopetrosis structurally closely resembled the induced bone changes in the rat. It is concluded that an important mechanism in the production of osteopetrosis is an accentuated rhythm of bone change like that shown experimentally to be produced in these animals. It is emphasised that these changes are but part of a range of bone disorders associated with abnormalities of cycles of resorption and deposition of bone, the type of change differing with the nature of the cycles

Objectives

Salubrinal is a synthetic agent that elevates phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) and alleviates stress to the endoplasmic reticulum. Previously, we reported that in chondrocytes, Salubrinal attenuates expression and activity of matrix metalloproteinase 13 (MMP13) through downregulating nuclear factor kappa B (NFκB) signalling. We herein examine whether Salubrinal prevents the degradation of articular cartilage in a mouse model of osteoarthritis (OA).

The effect of tenotomy and of immobilisation in lengthened and shortened positions on the intramuscular connective tissue of the calf muscles of the rat was studied morphometrically and with a scanning electron microscope. Both tenotomy and immobilisation produced a marked increase in both the endomysial and the perimysial collagen networks, with a simultaneous decrease in intramuscular capillary density. The increase in connective tissue volume was more pronounced and occurred more rapidly in the soleus, which consists mainly of type I, slow-twitch fibres than in the gastrocnemius, which is mainly of type II, fast-twitch fibres. The relative volume of connective tissue increased in parallel with the duration of immobilisation or after tenotomy. There was slightly more increase after immobilisation in a shortened rather than in a lengthened position

Fracture repair occurs by two broad mechanisms: direct healing, and indirect healing with callus formation. The effects of bisphosphonates on fracture repair have been assessed only in models of indirect fracture healing.

A rodent model of rigid compression plate fixation of a standardised tibial osteotomy was used. Ten skeletally mature Sprague–Dawley rats received daily subcutaneous injections of 1 µg/kg ibandronate (IBAN) and ten control rats received saline (control). Three weeks later a tibial osteotomy was rigidly fixed with compression plating. Six weeks later the animals were killed. Fracture repair was assessed with mechanical testing, radiographs and histology.

The mean stress at failure in a four-point bending test was significantly lower in the IBAN group compared with controls (8.69 Nmm^-2 (sd 7.63) vs 24.65 Nmm^-2 (sd 6.15); p = 0.017). On contact radiographs of the extricated tibiae the mean bone density assessment at the osteotomy site was lower in the IBAN group than in controls (3.7 mmAl (sd 0.75) vs 4.6 mmAl (sd 0.57); p = 0.01). In addition, histological analysis revealed progression to fracture union in the controls but impaired fracture healing in the IBAN group, with predominantly cartilage-like and undifferentiated mesenchymal tissue (p = 0.007).

Bisphosphonate treatment in a therapeutic dose, as used for risk reduction in fragility fractures, had an inhibitory effect on direct fracture healing. We propose that bisphosphonate therapy not be commenced until after the fracture has united if the fracture has been rigidly fixed and is undergoing direct osteonal healing.

Cite this article: Bone Joint J 2013;95-B:1263–8.

Bone marrow mesenchymal stromal cells were aspirated from immature male green fluorescent protein transgenic rats and cultured in a monolayer. Four weeks after the creation of the osteochondral defect, the rats were divided into three groups of 18: the control group, treated with an intra-articular injection of phosphate-buffered saline only; the drilling group, treated with an intra-articular injection of phosphate-buffered saline with a bone marrow-stimulating procedure; and the bone marrow mesenchymal stromal cells group, treated with an intra-articular injection of bone marrow mesenchymal stromal cells plus a bone marrow-stimulating procedure. The rats were then killed at 4, 8 and 12 weeks after treatment and examined.

The histological scores were significantly better in the bone marrow mesenchymal stromal cells group than in the control and drilling groups at all time points (p < 0.05). The fluorescence of the green fluorescent protein-positive cells could be observed in specimens four weeks after treatment.

Soaking bone grafts in a bisphosphonate solution before implantation can prevent their resorption and increase the local bone density in rats and humans. However, recent studies suggest that pre-treatment of allografts with bisphosphonate can prevent bone ingrowth into impaction grafts. We tested the hypothesis that excessive amounts of bisphosphonate would also cause a negative response in less dense grafts. We used a model where non-impacted metaphyseal bone grafts were randomised into three groups with either no bisphosphonate, alendronate followed by rinsing, and alendronate without subsequent rinsing, and inserted into bone chambers in rats. The specimens were evaluated histologically at one week, and by histomorphometry and radiology at four weeks. At four weeks, both bisphosphonate groups showed an increase in the total bone content, increased newly formed bone, and higher radiodensity than the controls. In spite of being implanted in a chamber with a limited opportunity to diffuse, even an excessive amount of bisphosphonate improved the outcome. We suggest that the negative results seen by others could be due to the combination of densely compacted bone and a bisphosphonate.

We suggest that bisphosphonates are likely to have a negative influence where resorption is a prerequisite to create space for new bone ingrowth.

The nervous system is known to be involved in inflammation and repair. We aimed to determine the effect of physical activity on the healing of a muscle injury and to examine the pattern of innervation. Using a drop-ball technique, a contusion was produced in the gastrocnemius in 20 rats. In ten the limb was immobilised in a plaster cast and the remaining ten had mobilisation on a running wheel. The muscle and the corresponding dorsal-root ganglia were studied by histological and immunohistochemical methods.

In the mobilisation group, there was a significant reduction in lymphocytes (p = 0.016), macrophages (p = 0.008) and myotubules (p = 0.008) between three and 21 days. The formation of myotubules and the density of nerve fibres was significantly higher (both p = 0.016) compared with those in the immobilisation group at three days, while the density of CGRP-positive fibres was significantly lower (p = 0.016) after 21 days.

Mobilisation after contusional injury to the muscle resulted in early and increased formation of myotubules, early nerve regeneration and progressive reduction in inflammation, suggesting that it promoted a better healing response.

In vitro studies on isolated bone cells were undertaken to investigate the presence of transplantation (histocompatibility) antigens. Bone cells were cultured with allogeneic lymphocytes and exposed to cytotoxic sera containing antibodies against transplantation antigens, to determine their antigenic profile. Preliminary results suggest that bone cells may not express lymphocyte stimulating antigens in an active form, at least after the isolation procedure performed. On the other hand, bone cells were killed by cytotoxic antibodies in a specific way, providing evidence for the presence of serologically defined (SD) transplantation antigens on the cell surface. Additional studies with absorbed sera suggest "sharing" of histocompatibility antigens between bone cells and lymphocytes. The relevance of the surface antigens of bone cells to clinical fields such as bone allotransplantation, susceptibility to various orthopaedic diseases and skeletal sarcomata is discussed.

A computerised image analyser has been used experimentally to determine its possible usefulness in quantifying bone density. Rats were prepared for this purpose and ash weight/volume readings were obtained on femora which had been radiographed and studied to estimate radiographic density by means of the image analyser. The findings indicated that rats can be prepared by administering oestradiol or by allowing an additional week's growth, both of which give a statistically significant increase of bone density. The computerised image analyser was able to detect these differences but the correlations, although real and linear, between ash weight/volume and radiographic density were not large. Further refinements in techniques will be needed before the system is applied clinically.

1. The effects of frozen irradiated homogenous bone grafts on the healing of circumscribed defects in the mandibles and femora of forty albino rats have been studied.

2. The grafts were well tolerated by the host animals and did not appear in any way either to stimulate or to inhibit osteogenesis. The rate of healing of the defects containing the homografts compared favourably with that of the control defects.

1. The effect of implanting heterogenous anorganic bone, homogenous organic bone, autogenous compact bone from the iliac crest, and autogenous bony callus into circumscribed defects in the femur of albino rats of the Wistar strain is described.

2. Neither heterogenous anorganic bone nor homogenous organic bone appeared to induce new bone formation in a healing defect.

3. Some of the osteogenic cells of autogenous callus implants survived transplantation to a bone defect and gave rise to new bone formation. This did not occur when compact bone from autogenous iliac crest was implanted.

4. Implants of autogenous callus, autogenous compact bone, homogenous organic bone and heterogenous anorganic bone all impeded the normal development of host bone trabeculae in a healing bone defect, seemingly because they acted as physical barriers to the proliferating host callus. None of the implant materials appeared to suppress the healing reaction ofthe host.

5. Implanted homogenous organic bone was removed and replaced by host bone more quickly than was implanted heterogenous anorganic bone, and it appears to be the better material for grafting into bone defects.

6. Autogenous callus or autogenous cancellous bone is a superior implant material to autogenous compact bone and is the bone graft material of choice.

7. The absorption of all the implant materials used in this investigation was associated with the presence of multinucleated giant cells.

8. The activity of multinucleated giant cells may be influenced by the organic matrix of the material which is to be absorbed.

9. Except when fresh autogenous callus was implanted into the defects, the rate of healing in the grafted defects was slower than that in the control defects. In the defects grafted with fresh autogenous callus the healing rates of the control and grafted defects were the same.