Rema Oliver

Relationship between age, skeletal site, and time post-ovariectomy on bone mineral and trabecular microarchitecture in rats

The ovariectomized (OVX) rat is widely used in osteoporosis research, but no standard model exists. The individual effects of rat age, skeletal site, and time post‐ovariectomy (post‐OVX) on bone have been examined. However, the relationship between them is not yet fully explored. This study examined how various combinations of rat age, skeletal site, and time post‐OVX affect bone mineral and microarchitecture. The rats used were 12 (n = 28), 24 (n = 28), and 44 (n = 31) weeks old. In each age group, approximately half underwent OVX and other half underwent Sham surgeries. Bone mineral (content and density) and trabecular morphology was assessed at 2, 5, 10, 15, 20, 25, and 30 weeks post‐surgery. Sites examined included the proximal tibia, spine, distal femur, and proximal femur. Overall, the proximal tibia showed the earliest and greatest differences between OVX and Sham groups. The 24‐week‐old group showed the best osteoporotic response. The 12‐week‐old group showed growth effects, whilst the 44‐week‐old group showed aging effects. The response of certain sites to OVX was also found to depend on the rat age used. These findings may aid in explaining discrepancies reported in the literature as well as synergistic combinations that may signify advanced conditions.

The Masquelet Technique for Membrane Induction and the Healing of Ovine Critical Sized Segmental Defects

The healing of critical sized segmental defects is an ongoing clinical problem. No method has achieved pre-eminence. The Masquelet technique is a relatively new innovation involving the induction of a fibrous tissue membrane around the bone defect site taking advantage of the body’s foreign body reaction to the presence of a polymethylmethacrylate (PMMA) spacer. The aim of this study was to investigate the properties and characteristics of this induced membrane and its effectiveness when used in conjunction with allograft or an allograft/autograft mix as filler materials in an ovine critical sized defect model. The resultant induced membrane was found to be effective in containing the graft materials in situ. It was demonstrated to be an organised pseudosynovial membrane which expressed bone morphogenic protein 2 (BMP2), transforming growth factor- beta (TGFβ), vascular endothelial growth factor (VEGF), von Willerbrand factor (vWF), interleukin 6 (IL-6) and interleukin 8 (IL-8). While more new bone growth was evident in the test groups compared to the controls animals at 12 weeks, the volumes were not statistically different and no defects were fully bridged. Of the two graft material groups, the allograft/autograft mix was shown to have a more rapid graft resorption rate than the allograft only group. While the Masquelet technique proved effective in producing a membrane to enclose graft materials, its ability to assist in the healing of critical sized segmental defects when compared to empty controls remained inconclusive.

Effects of Demineralized Bone Matrix on Tendon-Bone Healing in an Intra-articular Rodent Model

Background: Techniques to improve and accelerate tendon-bone healing could be advantageous in anterior cruciate ligament (ACL) reconstruction. Effects of demineralized bone matrix (DBM) on intra-articular tendon-bone healing have not been examined. Hypothesis: Demineralized bone matrix has the potential to convey osteoinductive growth proteins to the site of healing at the tendon-bone interface. We hypothesized that the presence of DBM will result in more bone formation and hasten tendon-bone healing. Study Design: Controlled laboratory study. Methods: Fifty-six female athymic rnu/rnu (nude) rats were used. Rats were randomly allocated into 2 groups (control or treatment). The control group underwent an ACL reconstruction, while the treatment group had human DBM implanted in the tendon graft and bone tunnel before reconstruction. Rats were sacrificed at 2 (n = 8), 4 (n = 24), and 6 (n = 24) weeks for histological, and immunohistochemical (t = 2, 4, and 6 weeks), and biomechanical testing and micro–computed tomography (t = 4 and 6 weeks) end points. Results: Our findings suggest that in the presence of DBM, tendon-bone healing is augmented by increased woven bone formation and enhanced bone remodeling as indicated by histology and micro–computed tomography. This ultimately resulted in a statistically significant increase in peak load to failure of the tendon-bone interface at 4 weeks (DBM group: 5.96 ± 1.36 N; control group: 2.86 ± 0.7 N) and 6 weeks (DBM group: 9.13 ± 0.97 N; control group: 5.81 ± 1.1 N). Conclusion: Demineralized bone matrix at the tendon-bone interface promotes healing between the tendon and bone in a rodent ACL model. Clinical Relevance: Introduction of osteoinductive DBM at the tendon-bone interface during ACL reconstructive surgery may improve short-term outcomes.

Posterolateral spinal fusion in a rabbit model using a collagen–mineral composite bone graft substitute

Choosing the appropriate graft material to participate in the healing process in posterolateral spinal fusion continues to be a challenge. Combining synthetic graft materials with bone marrow aspirate (BMA) and autograft is a reasonable treatment option for surgeons to potentially reduce or replace the need for autograft. FormaGraft, a bone graft material comprising 12% bovine-derived collagen and 88% ceramic in the form of hydroxyapatite (HAp) and beta tricalcium phosphate (β-TCP) was evaluated in three possible treatment modalities for posterior spinal fusion in a standard rabbit model. These three treatment groups were FormaGraft alone, FormaGraft soaked in autogenous BMA, and FormaGraft with BMA and iliac crest autograft. No statistically demonstrable benefits or adverse effects of the addition of BMA were found in the current study based on macroscopic, radiology or mechanical data. This may reflect, in part, the good to excellent results of the collagen HA/TCP composite material alone in a well healing bony bed. Histology did, however, reveal a benefit with the use of BMA. Combining FormaGraft with autograft and BMA achieved results equivalent to autograft alone. The mineral and organic nature of the material provided a material that facilitated fusion between the transverse processes in a standard preclinical posterolateral fusion model.

PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells

Significance In this report we describe the generation of tissue-regenerative multipotent stem cells (iMS cells) by treating mature bone and fat cells transiently with a growth factor [platelet-derived growth factor–AB (PDGF-AB)] and 5-Azacytidine, a demethylating compound that is widely used in clinical practice. Unlike primary mesenchymal stem cells, which are used with little objective evidence in clinical practice to promote tissue repair, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner without forming tumors. This method can be applied to both mouse and human somatic cells to generate multipotent stem cells and has the potential to transform current approaches in regenerative medicine. Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor–AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

Platelet-rich plasma and bone defect healing.

On activation, platelets secrete an array of growth factors that contribute to bone regeneration. Combining platelet-rich plasma (PRP) with bone graft substitutes has the potential to reduce or replace the reliance on autografts. Lack of standardization and improper use may contribute to the conflicting outcomes reported within both preclinical and clinical investigations using PRP. This study investigates the effect of PRP dose on bone augmentation. Eighty critical-sized defects were created in the cancellous bone of the medial proximal tibia and the distal femur of 20 five-year-old female sheep. The defects were treated with three doses of an autologous thrombin-activated PRP combined with a biphasic calcium phosphate (BCP) or autograft and empty defects. Radiography, micro-computed tomography, histology, histomorphometry, and fluorochrome bone labels were examined at 4 weeks. The empty defects did not spontaneously heal. The highest dose of PRP treatment had a significantly greater micro-CT bone volume/total volume compared with the BCP alone (PRP: 30.6%±1.8%; BCP: 24.5%±0.1%). All doses of PRP treatment were significantly greater than the BCP alone for histomorphometric new bone area (PRP: 14.5%±1.3%; BCP: 9.7%±1.5%) and bone ingrowth depth (PRP: 2288±210 μm; BCP:1151±268 μm). From week 2 onward, PRP had a significant effect on the weekly bone ingrowth compared with BCP; however, autografts had the highest amount of weekly fluorescent bone labeling. PRP induces new bone formation with a dose-dependent response at 4 weeks when used with a BCP in sheep.

The histology of facial aesthetic subunits: Implications for common nasal reconstructive procedures

Reconstruction of cutaneous nasal defects is often a challenging problem with multiple solutions. Many factors must be considered when deciding on the appropriate reconstructive procedure, including optimally matching donor site skin to the original recipient site skin. To the best of our knowledge no objective study has been undertaken to examine which areas best match the histological features of skin from various nasal cosmetic subunits. We have undertaken a descriptive histological analysis of skin from 25 facial and nasal aesthetic subunits from four male Caucasian cadavers, aged 65-88. The three variables looked at were epidermal thickness, dermal thickness and density of pilosebaceous subunits. Our findings have been plotted on photographs of the face to provide visual maps of facial histological features by cosmetic subunit. Our results show that histologically, the best matched skin for reconstructing a given nasal defect is likely to come from an adjacent nasal subunit. Looking at distant donor sites, the helical root, helical rim and pre-auricular area are closest to nasal skin in terms of dermal thickness. In terms of density of pilosebaceous units, the helical root, pre-auricular area and lateral forehead are the three areas best matched to nasal skin.

Fibulin-1 Predicts Disease Progression in Patients With Idiopathic Pulmonary Fibrosis

BACKGROUND The underlying mechanisms of idiopathic pulmonary fibrosis (IPF) are unknown. This progressive disease has high mortality rates, and current models for prediction of mortality have limited value in identifying which patients will progress. We previously showed that the glycoprotein fibulin-1 is involved in enhanced proliferation and wound repair by mesenchymal cells and, thus, may contribute to lung fibrosis in IPF. METHODS Serum, lung tissue, and lung function values were obtained from four independent locations (Sydney, NSW, and Perth, WA, Australia; San Francisco, CA; and Modena, Italy). Patients with IPF were followed for a minimum of 1 year and progression was defined as a significant decline in lung function or death. Primary parenchymal lung fibroblasts of 15 patients with and without IPF were cultured under nonstimulatory conditions. Fibulin-1 levels in serum, and secreted or deposited by fibroblasts, were measured by western blot and in lung tissue by immunohistochemistry. RESULTS Serum fibulin-1 levels were increased in patients with IPF compared with subjects without lung disease (P = .006). Furthermore, tissue fibulin-1 levels were increased in patients with IPF (P = .02) and correlated negatively with lung function (r = -0.9, P < .05). Primary parenchymal fibroblasts from patients with IPF produced more fibulin-1 than those from subjects without IPF (P < .05). Finally, serum fibulin-1 levels at first blood draw predicted disease progression in IPF within 1 year (area under the curve , 0.71; 95% CI, 0.57-0.86; P = .012). CONCLUSIONS Fibulin-1 is a novel potential biomarker for disease progression in IPF and raises the possibility that it could be used as a target for the development of new treatments.

Transdermal fentanyl and its use in ovine surgery

Fentanyl delivered via a transdermal patch has the potential to decrease the need for post-operative handling of sheep undergoing surgical procedures. Two studies were performed to test: (1) the ideal timing for the application of pre-emptive analgesic patches and (2) the efficacy of a 2 µg/kg/h dose, as extrapolated from other species. The first study had sheep divided into two groups. Group 1 had a fentanyl patch applied for 24 h prior to a patch change and group 2 had a fentanyl patch applied 72 h prior to a change. The second study applied the results obtained in the first and tested the efficacy of 2 µg/kg/h as an effective dose in an orthopaedic surgical environment. Results indicated that the ideal time for pre-emptive fentanyl patch administration is 24-36 h prior to surgery and that 2 µg/kg/h is an effective minimum therapeutic dose rate for the use of fentanyl as an analgesic in an orthopaedic surgical environment.

Development of a Novel Model for the Assessment of Dead-Space Management in Soft Tissue

Following extensive surgical debridement in the treatment of infection, a “dead space” can result following surgical closure that can fill with hematoma, an environment conducive to bacterial growth. The eradication of dead space is essential in order to prevent recurrent infection. This study describes a novel small animal model to investigate dead-space management in muscle tissue. Two absorbable test materials were implanted in each animal; beads of calcium sulfate alone, and beads loaded with vancomycin and tobramycin. In-life blood samples and radiographs were taken from each animal following implantation. Animals were sacrificed at 1, 7, 21, 42, and 63 days post-operatively (n = 4), and implant sites were analysed by micro-computed tomography, histology and immunohistochemistry. Complete resorption was confirmed radiographically at 3 weeks post-implantation. Histologically, the host tissue response to both materials was identical, and subsequent healing at the implant sites was observed with no dead space remaining. Vancomycin was not detected in blood serum. However, peak tobramycin levels were detected in all animals at 6 hours post-implantation with no detectable levels in any animals at 72 hours post implantation. Serological inflammatory cytokine expression for IL-6, TNF-α and IL-1β indicated no unusual inflammatory response to the implanted materials or surgical procedure. The model was found to be convenient and effective for the assessment of implant materials for management of dead space in muscle tissue. The two materials tested were effective in resolving the surgically created dead space, and did not elicit any unexpected adverse host response.