Developing a Tooth in situ Organ Culture Model for Dental and Periodontal Regeneration Research.

In this study we have realized the need for an organ culture tooth in situ model to simulate the tooth structure especially the tooth attachment apparatus. The importance of such a model is to open avenues for investigating regeneration of the complex tooth and tooth attachment tissues and to reduce the need for experimental animals in investigating dental materials and treatments in the future. The aim of this study was to develop a porcine tooth in situ organ culture model and a novel bioreactor suitable for future studies of periodontal regeneration, including application of appropriate physiological loading. The Objectives of this study was to establish tissue viability, maintenance of tissue structure, and model sterility after 1 and 4 days of culture. To model diffusion characteristics within the organ culture system and design and develop a bioreactor that allows tooth loading and simulation of the chewing cycle. Methods: Twenty-one porcine first molars were dissected aseptically in situ within their bony sockets. Twelve were used to optimize sterility and determine tissue viability. The remainder were used in a 4-day organ culture study in basal medium. Sterility was determined for medium samples and swabs taken from all tissue components, using standard aerobic and anaerobic microbiological cultures. Tissue viability was determined at days 1 and 4 using an XTT assay and Glucose consumption assays. Maintenance of structure was confirmed using histology and histomorphometric analysis. Diffusion characteristics were investigated using micro-CT combined with finite element modeling. A suitable bioreactor was designed to permit longer term culture with application of mechanical loading to the tooth in situ. Result: XTT and Glucose consumption assays confirmed viability throughout the culture period for all tissues investigated. Histological and histomorphometric analysis confirmed maintenance of tissue structure. Clear microbiological cultures indicated maintenance of sterility within the organ culture system. The novel bioreactor showed no evidence of medium contamination after 4 days of culture. Finite element modeling indicated nutrient availability to the periodontium. Conclusion: A whole tooth in situ organ culture system was successfully maintained over 4 days in vitro.

Surgical technique for lumbar intervertebral disc transplantation in a goat model.

PURPOSE: Fresh-frozen intervertebral disc transplantation was determined to be an effective treatment for degenerative disc diseases in rhesus monkeys and in humans. Further research in improving different aspects of disc allografts transplantation is needed and will be investigated in large animal models. This study reports the detailed surgical technique of intervertebral disc transplantation without internal fixation and the important notes to ensure success in goats. METHODS: Fifty-one male goats were used in this study. Ten goats were used as intervertebral disc allograft donors; the remaining forty-one goats were used to develop the surgical technique for intervertebral disc allograft transplantation. Radiographs, ex vivo MRI and gross observation were used to monitor the stability and healing of the disc allografts at 3 months, postoperatively. RESULTS: Size matching of the disc allograft, preservation of the anterior longitudinal ligament and an appropriate portion of the annulus fibrosus at the recipient site were crucial for stable graft retention. Additionally, a slightly reduced height of the disc allograft compared to that of the recipient slot may avoid graft endplate fracture. CONCLUSIONS: Lumbar intervertebral disc transplantation without internal fixation can be successfully performed in goats.

The effect of remodeling on the kinematics of the malpositioned disc allograft transplantation.

STUDY DESIGN: A postoperative biomechanical study. OBJECTIVE: This study aimed to assess whether the mal-alignment of the intervertebral disc (IVD) allograft during transplantation would negatively affect the biomechanics of the spinal segment. SUMMARY OF BACKGROUND DATA: Studies of human IVD allograft transplantation have observed remodeling of the allograft implant, suggesting that the remodeling of the allograft may be able to restore the natural mechanics of the IVD. METHODS: Eighteen male goats (age: 6-12 months; weight: 25-30 kg) were randomly assigned into control (n = 5), aligned (n = 5), or malpositioned (n = 5) groups. Transplantation of a size-matched cryopreserved IVD allograft was performed in the lumbar region (L4-L5) after disc excision. In the aligned group, the IVD allografts were placed aligned and flush with the anterior vertebral margin. In the malpositioned group, the allografts were placed proud anteriorly by 25% of the anterior-posterior diameter of the allograft. The lumbar spines were harvested at 6 months after transplantation. Three-dimensional kinematic assessment of the lumbar spines was performed using an MTS testing machine and an optoelectronic camera system. The range of motion, neutral zone, and instantaneous axis of rotation were calculated. RESULTS: No significant difference in range of motion was noted between the groups in flexion, axial rotation, and lateral bending. Significance was noted with extension range of motion as detected in both the aligned (17.51 ± 1.97 degrees; P = 0.019) and malpositioned groups (16.61 ± 2.35 degrees; P = 0.027) compared with the control (10.11 ± 1.03 degrees). No significant difference was detected in the neutral zone between the groups. Significant difference in the instantaneous axis of rotation orientation between the malpositioned and control groups was detected in the sagittal plane during lateral bending motion (P = 0.036). CONCLUSION: Kinematic parameters in both the aligned and malpositioned allograft were similar to those of the intact spine. This suggests that precise positioning of the IVD allograft may not be an essential factor affecting the biomechanics of the spinal segment after transplantation.

Osteoid osteoma of the femoral neck causes deformity in children: a case report.

Intra-articular osteoid osteoma (IAOO) is rare and non-specific both in clinical and radiographic presentations, thus often associated with delayed diagnosis. Synovitis and muscle atrophy occur in adult IAOO. Bony deformity can occur in children and has been ascribed to muscle contractures. We report a case of a child with IAOO in the femoral neck. There was an absence of muscle contracture during surgery, and therefore the femoral deformity may have been induced directly by IAOO in the early stage of the disease.

REST corepressor (CoREST) repression induces phenotypic gene regulation in advanced osteoarthritic chondrocytes.

Alternations in cartilage chondrocyte phenotype characteristic by the decreased type II collagen and aggrecan together with increased type X collagen synthesis serve as a beacon for osteoarthritis progression. However, little is known about the underlying molecular mechanisms. The current study seeks to discover molecules that involved in osteoarthritic chondrocytes phenotype regulation. Differential proteomics was generated with two-dimensional gel electrophoresis between normal articular cartilage (NAC) and advanced osteoarthritic cartilage (AOC). Those differentially expressed proteins were identified by mass spectrometry. The down-regulation of a neuronal silencer, the REST corepressor (CoREST) in AOC, was verified by Western blot. CoREST silencing was performed in primarily cultured NAC chondrocytes with specific siRNA to reveal the possible involvement of CoREST repression in chondrocyte phenotypic genes modulation. Ninteen differentially expressed proteins were screened and identified. Among these proteins, CoREST, HHL, and zinc finger protein 155 were estimated to be possible gene modulators. CoREST protein level was verified to be down-regulated by 69.5% (p < 0.001) in AOC. In response to CoREST knock-down by 64.8% (p < 0.001) in NAC chondrocytes, the gene expression level of the chondrocyte terminal differentiation marker gene, collagen X was found to be up-regulated by 40.0% (p = 0.017), whereas the chondrocyte differentiation phenotypic genes, collagen II and aggrecan were down-regulated by 71.4% (p < 0.001) and 57.6% (p < 0.001), respectively. The results indicate that the silencing of CoREST by siRNA transfection in NAC may reflect CoREST repression in AOC, which results in phenotypic genes modulation and suggests a homeostatic role of this transcription factor in articular chondrocyte.

Minimizing cryopreservation-induced loss of disc cell activity for storage of whole intervertebral discs.

Severe intervertebral disc (IVD) degeneration often requires disc excision and spinal fusion, which leads to loss of spinal segment mobility. Implantation of an allograft disc or tissue engineered disc construct emerges as an alternative to artificial disc replacement for preserving the motion of the degenerated level. Establishment of a bank of cadaveric or engineered cryopreserved discs enables size matching, and facilitates clinical management. However, there is a lack of understanding of the behaviour of disc cells during cryopreservation, as well as how to maximize their survival, such that disc graft properties can be preserved. Here, we report on the effect of alterations in cooling rates, cryoprotective agents (CPAs), and duration of pre-cryopreservation incubation in CPA on cellular activity in whole porcine lumbar discs. Our results indicated that cooling rates of -0.3 degrees C/min and -0.5 degrees C/min resulted in the least loss of metabolic activity in nucleus pulposus (NP) and annulus fibrosus (AF) respectively, while metabolic activity is best maintained by using a combination of 10% dimethylsulphoxide (DMSO) and 10% propylene-glycol (PG) as CPA. By the use of such parameters, metabolic activity of the NP and the AF cells could be maintained at 70% and 45%, respectively, of that of the fresh tissue. Mechanical testing and histological evaluation showed no significant differences in mechanical properties or alterations in disc structure compared to fresh discs. Despite the limitations of the animal model, our findings provide a framework for establishing an applicable cryopreservation protocol for human disc allografts or tissue-engineered disc constructs.