Biological performance of a polycaprolactone-based scaffold used as fusion cage device in a large animal model of spinal reconstructive surgery

A bioactive and bioresorbable scaffold fabricated from medical grade poly (epsilon-caprolactone) and incorporating 20% beta-tricalcium phosphate (mPCL–TCP) was recently developed for bone regeneration at load bearing sites. In the present study, we aimed to evaluate bone ingrowth into mPCL–TCP in a large animal model of lumbar interbody fusion. Six pigs underwent a 2-level (L3/4; L5/6) anterior lumbar interbody fusion (ALIF) implanted with mPCL–TCP þ 0.6 mg rhBMP-2 as treatment group while four other pigs implanted with autogenous bone graft served as control. Computed tomographic scanning and histology revealed complete defect bridging in all (100%) specimen from the treatment group as early as 3 months. Histological evidence of continuing bone remodeling and maturation was observed at 6 months. In the control group, only partial bridging was observed at 3 months and only 50% of segments in this group showed complete defect bridging at 6 months. Furthermore, 25% of segments in the control group showed evidence of graft fracture, resorption and pseudoarthrosis. In contrast, no evidence of graft fractures, pseudoarthrosis or foreign body reaction was observed in the treatment group. These results reveal that mPCL–TCP scaffolds could act as bone graft substitutes by providing a suitable environment for bone regeneration in a dynamic load bearing setting such as in a porcine model of interbody spine fusion.

The evaluation of a biphasic osteochondral implant coupled with an electrospun membrane in a large animal model

Conventional clinical therapies are unable to resolve osteochondral defects adequately, hence tissue engineering solutions are sought to address the challenge. A biphasic implant which was seeded with Mesenchymal Stem Cells (MSC) and coupled with an electrospun membrane was evaluated as an alternative. This dual phase construct comprised of a Polycaprolactone (PCL) cartilage scaffold and a Polycaprolactone - Tri Calcium Phosphate (PCL - TCP) osseous matrix. Autologous MSC was seeded into the entire implant via fibrin and the construct was inserted into critically sized osteochondral defects located at the medial condyle and patellar groove of pigs. The defect was resurfaced with a PCL - collagen electrospun mesh that served as a substitute for periosteal flap in preventing cell leakage. Controls either without implanted MSC or resurfacing membrane were included. After 6 months, cartilaginous repair was observed with a low occurrence of fibrocartilage at the medial condyle. Osteochondral repair was promoted and host cartilage degeneration was arrested as shown by the superior Glycosaminoglycan (GAG) maintenance. This positive morphological outcome was supported by a higher relative Young's modulus which indicated functional cartilage restoration. Bone in growth and remodeling occurred in all groups with a higher degree of mineralization in the experimental group. Tissue repair was compromised in the absence of the implanted cells or the resurfacing membrane. Moreover healing was inferior at the patellar groove as compared to the medial condyle and this was attributed to the native biomechanical features.

Inhibition of p38 pathway leads to OA-like changes in a rat animal model

Objectives The p38 mitogen-activated protein kinase (MAPK) signal transduction pathway is involved in a variety of inflammatory responses, including cytokine generation, cell differentiation proliferation and apoptosis. Here, we examined the effects of systemic p38 MAPK inhibition on cartilage cells and osteoarthritis (OA) disease progression by both in vitro and in vivo approaches. Methods p38 kinase activity was evaluated in normal and OA cartilage cells by measuring the amount of phosphorylated protein. To examine the function of p38 signaling pathway in vitro, normal chondrocytes were isolated and differentiated in the presence or absence of p38 inhibitor; SB203580 and analysed for chondrogenic phenotype. Effect of systemic p38 MAPK inhibition in normal and OA (induced by menisectomy) rats were analysed by treating animals with vehicle alone (DMS0) or p38 inhibitor (SB203580). Damage to the femur and tibial plateau was evaluated by modified Mankin score, histology and immunohistochemistry. Results Our in vitro studies have revealed that a down-regulation of chondrogenic and increase of hypertrophic gene expression occurs in the normal chondrocytes, when p38 is neutralized by a pharmacological inhibitor. We further observed that the basal levels of p38 phosphorylation were decreased in OA chondrocytes compared with normal chondrocytes. These findings together indicate the importance of this pathway in the regulation of cartilage physiology and its relevance to OA pathogenesis. At in vivo level, systematic administration of a specific p38 MAPK inhibitor, SB203580, continuously for over a month led to a significant loss of proteoglycan; aggrecan and cartilage thickness. On the other hand, SB203580 treated normal rats showed a significant increase in TUNEL positive cells, cartilage hypertrophy markers such as Type 10 collagen, Runt-related transcription factor and Matrix metalloproteinase-13 and substantially induced OA like phenotypic changes in the normal rats. In addition, menisectomy induced OA rat models that were treated with p38 inhibitor showed aggravation of cartilage damage. Conclusions In summary, this study has provided evidence that the component of the p38 MAPK pathway is important to maintain the cartilage health and its inhibition can lead to severe cartilage degenerative changes. The observations in this study highlight the possibility of using activators of the p38 pathway as an alternative approach in the treatment of OA.

Tissue engineering bone - reconstruction of critical sized segmental bone defects in a large animal model

Currently, well established clinical therapeutic approaches for bone reconstruction are restricted to the transplantation of autografts and allografts, and the implantation of metal devices or ceramic-based implants to assist bone regeneration. Bone grafts possess osteoconductive and osteoinductive properties, their application, however, is associated with disadvantages. These include limited access and availability, donor site morbidity and haemorrhage, increased risk of infection, and insufficient transplant integration. As a result, recent research focuses on the development of complementary therapeutic concepts. The field of tissue engineering has emerged as an important alternative approach to bone regeneration. Tissue engineering unites aspects of cellular biology, biomechanical engineering, biomaterial sciences and trauma and orthopaedic surgery. To obtain approval by regulatory bodies for these novel therapeutic concepts the level of therapeutic benefit must be demonstrated rigorously in well characterized, clinically relevant animal models. Therefore, in this PhD project, a reproducible and clinically relevant, ovine, critically sized, high load bearing, tibial defect model was established and characterized as a prerequisite to assess the regenerative potential of a novel treatment concept in vivo involving a medical grade polycaprolactone and tricalciumphosphate based composite scaffold and recombinant human bone morphogenetic proteins.

Quantitative and qualitative assessment of the regenerative potential of osteoblasts versus bone marrow derived mesenchymal stem cells in the reconstruction of critical sized segmental tibial bone defects in a large animal model

This thesis is about the use of different cells for bone tissue engineering. The cells were used in combination with a novel biomaterial in a large tibial bone defects in a sheep model. Furthermore this study developed a novel cell delivery procedure for bone tissue engineering. This novel procedure of cell delivery could overcome the current problems of cell-based tissue engineering and serve as a baseline for the translation of novel concepts into clinical application.

Longitudinal performance of polycaprolactone-based scaffold plus recombinant human morphogenetic protein-2 (rhBMP-2) in large preclinical animal model : 6- versus 12 months

There is strong current interest in the use of biodegradable scaffolds in combination with bone growth factors as a valuable alternative to the current gold standard autograft in spinal fusion surgery Yong et al. (2013). Here we report on 6- vs 12- month data set evaluating the longitudinal performance of a CaP coated polycaprolactone (PCL) scaffold loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) as a bone graft substitute within a preclinical ovine thoracic spine. The results of this study demonstrate the efficacy of scaffold-based delivery of rhBMP-2 in promoting higher fusion grades at 6- and 12- months in comparison to the scaffold alone or autograft group within the same time frame. Fusion grades achieved at six months using PCL+rhBMP-2 are not significantly increased at twelve months post surgery.

Animal model and molecular interactions of Cln5

Neuronal ceroid lipofuscinoses (NCLs) are a family of inherited pediatric neurodegenerative disorders, leading to retinal degeneration, death of selective neuronal populations and accumulation of autofluorscent ceroid-lipopigments. The clinical manifestations are generally similar in all forms. The Finnish variant late infantile neuronal ceroid lipofuscinosis (vLINCLFin) is a form of NCL, especially enriched in the Finnish population. The aim of this thesis was to analyse the brain pathology of vLINCLFin utilising the novel Cln5-/- mouse model. Gene expression profiling of the brains of already symptomatic Cln5-/- mice revealed that inflammation, neurodegeneration and defects in myelinization are the major characteristics of the later stages of the disease. Histological characterization of the brain pathology confirmed that the thalamocortical system is affected in Cln5-/- mice, similarly to the other NCL mouse models. However, whereas the brain pathology in all other analyzed NCL mice initiate in the thalamus and spread only months later to the cortex, we observed that the sequence of events is uniquely reversed in Cln5-/- mice; beginning in the cortex and spreading to the thalamus only months later. We could also show that even though neurodegeneration is inititated in the cortex, reactive gliosis and loss of myelin are evident in specific nuclei of the thalamus already in the 1 month old brain. To obtain a deeper insight into the disturbed metabolic pathways, we performed gene expression profiling of presymptomatic mouse brains. We validated these findings with immunohistological analyses, and could show that cytoskeleton and myelin were affected in Cln5-/- mice. Comparison of gene expression profiling results of Cln5-/- and Cln1-/- mice, further highlighted that these two NCL models share a common defective pathway, leading to disturbances in the neuronal growth cone and cytoskeleton. Encouraged by the evidence of this defected pathway, we analyzed the molecular interactions of NCL-proteins and observed that Cln5 and Cln1/Ppt1 proteins interact with each other. Furthermore, we demonstrated that Cln5 and Cln1/Ppt1 share an interaction partner, the F1-ATP synthase, potentially linking both vLINCLFIN and INCL diseases to disturbed lipid metabolism. In addition, Cln5 was shown to interact with other NCL proteins; Cln2, Cln3, Cln6 and Cln8, implicating a central role for Cln5 in the NCL pathophysiology. This study is the first to describe the brain pathology and gene expression changes in the Cln5-/- mouse. Together the findings presented in this thesis represent novel information of the disease processes and the molecular mechanisms behind vLINCLFin and have highlighted that vLINCLFin forms a very important model to analyze the pathophysiology of NCL diseases.

Probiotics modify tight junction proteins in an animal model of non-alcoholic fatty liver disease

Objectives We have investigated the effects of a multi–species probiotic preparation containing a combination of probiotic bacterial genera that included Bifidobacteria, Lactobacilli and a Streptococcus in a mouse model of high fat diet/obesity induced liver steatosis. Methods Three groups of C57B1/6J mice were fed either a standard chow or a high fat diet for 20 weeks, while a third group was fed a high fat diet for 10 weeks and then concomitantly administered probiotics for a further 10 weeks. Serum, liver and large bowel samples were collected for analysis. Results The expression of the tight junction proteins ZO-1 and ZO-2 was reduced (p < 0.05) in high fat diet fed mice compared to chow fed mice. Probiotic supplementation helped to maintain tight ZO-1 and ZO-2 expression compared with the high fat diet group (p < 0.05), but did not restore ZO-1 or ZO-2 expression compared with chow fed mice. Mice fed a high fat diet ± probiotics had significant steatosis development compared to chow fed mice (p < 0.05); steatosis was less severe in the probiotics group compared to the high fat diet group. Hepatic triglycerides concentration was higher in mice fed a high fat diet ± probiotics compared to the chow group (p < 0.05), and was lower in the probiotics group compared to the high fat diet group (p < 0.05). Compared to chow fed mice, serum glucose and cholesterol concentrations, and the activity of alanine transaminase were higher (p < 0.05), whereas serum triglyceride concentration was lower (p < 0.05) in mice fed a high fat diet ± probiotics. Conclusions Supplementation with a multi-species probiotic formulation helped to maintain tight junction proteins ZO-1 and ZO-2, and reduced hepatic triglyceride concentrations compared with a HFD alone.

Better early osteogenesis of electroconductive hydroxyapatite-calcium titanate composites in a rabbit animal model

In view of the fact that bone healing can be enhanced due to external electric field application, it is important to assess the influence of the implant conductivity on the bone regeneration in vivo. To address this issue, this study reports the in vivo biocompatibility property of multistage spark plasma sintered hydroxyapatite (HA)-80 wt % calcium titanate (CaTiO3) composites and monolithic HA, which have widely different conductivity property (14 orders of magnitude difference). The ability of bone regeneration was assessed by implantation in cylindrical femoral bone defects of rabbit animal model for varying time period of 1, 4, and 12 weeks. The overall assessment of the histology results suggests that the progressive healing of bone defects around HA-80 wt % CaTiO3 is associated with a better efficacy with respect to (w.r.t) early stage neobone formation, which is histomorphometrically around 140% higher than monolithic HA. Overall, this study demonstrates that the in vivo biocompatibility property of HA-80 wt % CaTiO3 with respect to local effects after 12 weeks of implantation is not compromised both qualitatively and quantitatively, and a comparison with control implant (HA) points toward the critical role of electrical conductivity on better early stage bone regeneration. (c) 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 842-851, 2014.

In vitro osteogenic cell proliferation, mineralization, and in vivo osseointegration of injection molded high-density polyethylene-based hybrid composites in rabbit animal model

The present work reports the biocompatibility property of injection molded HDPE-HA-Al2O3 hybrid composites. In vitro cytocompatibility results reveal that osteogenic cell viability and bone mineralization are favorably supported in a statistically significant manner on HDPE-20% HA-20% Al2O3 composite, in comparison to HDPE-40 wt.% HA or HDPE-40 wt.% Al2O3. The difference in cytocompatibility property is explained in terms of difference in substrate wettability/surface energy and importantly, both the cell proliferation at 7 days or bone mineralization at 21 days on HDPE-20% HA-20% Al2O3 composite are either comparable or better than sintered HA. The progressive healing of cylindrical femoral bone defects in rabbit animal model was assessed by implantation experiments over 1, 4 and 12 weeks. Based on the histological analysis as well as histomorphometrical evaluation, a better efficacy of HDPE-20% HA-20% Al2O3 over high-density polyethylene (HDPE) for bone regeneration and neobone formation at host bone-implant interface was established. Taken together, the present study unequivocally establishes that despite the presence of 20% Al2O3, HDPE-based hybrid composites are as biocompatible as HA in vitro or better than HDPE in vivo.

Development, in vitro and in vivo characterization of zoledronic acid functionalized hydroxyapatite nanoparticle based formulation for treatment of osteoporosis in animal model

We investigated the potential of using novel zoledronic acid (ZOL)-hydroxyapatite (HA) nanoparticle based drug formulation in a rat model of postmenopausal osteoporosis. By a classical adsorption method, nanoparticles of HA loaded with ZOL (HNLZ) drug formulation with a size range of 100-130 nm were prepared. 56 female Wistar rats were ovariectomized (OVX) or sham-operated at 3 months of age. Twelve weeks post surgery, rats were randomized into seven groups and treated with various doses of HNLZ (100, 50 and 25 mu g/kg, intravenous single dose), ZOL (100 mu g/kg, intravenous single dose) and HA nanoparticle (100 mu g/kg, intravenous single dose). Untreated OVX and sham OVX served as controls. After three months treatment period, we evaluated the mechanical properties of the lumbar vertebra and femoral mid-shaft. Femurs were also tested for trabecular microarchitecture. Sensitive biochemical markers of bone formation and bone resorption in serum were also determined. With respect to improvement in the mechanical strength of the lumbar spine and the femoral mid-shaft, the therapy with HNLZ drug formulation was more effective than ZOL therapy in OVX rats. Moreover, HNLZ drug therapy preserved the trabecular microarchitecture better than ZOL therapy in OVX rats. Furthermore, the HNLZ drug formulation corrected increase in serum levels of bone-specific alkaline phosphatase, procollagen type I N-terminal propeptide, osteocalcin, tartrate-resistant acid phosphatase 5b and C-telopeptide of type 1 collagen better than ZOL therapy in OVX rats. The results strongly suggest that HNLZ novel drug formulation appears to be more effective approach for treating severe osteoporosis in humans. (C) 2014 Elsevier B.V. All rights reserved.