Utilizing micro-computed tomography to evaluate bone structure surrounding dental implants: a comparison with histomorphometry

Although histology has proven to be a reliable method to evaluate the ossoeintegration of a dental implant, it is costly, time consuming, destructive, and limited to one or few sections. Microcomputed tomography (µCT) is fast and delivers three-dimensional information, but this technique has not been widely used and validated for histomorphometric parameters yet. This study compared µCT and histomorphometry by means of evaluating their accuracy in determining the bone response to two different implant materials. In total, 32 titanium (Ti) and 16 hydroxyapatite (HA) implants were installed in 16 lop-eared rabbits. After 2 and 4 weeks, the animals were scarified, and the samples retrieved. After embedding, the samples were scanned with µCT and analyzed three-dimensionally for bone area (BA) and bone-implant contact (BIC). Thereafter, all samples were sectioned and stained for histomorphometry. For the Ti implants, the mean BIC was 25.25 and 28.86% after 2 and 4 weeks, respectively, when measured by histomorphometry, while it was 24.11 and 24.53% when measured with µCT. BA was 35.4 and 31.97% after 2 and 4 weeks for histomorphometry and 29.06 and 27.65% for µCT. For the HA implants, the mean BIC was 28.49 and 42.51% after 2 and 4 weeks, respectively, when measured by histomorphometry, while it was 33.74 and 42.19% when measured with µCT. BA was 30.59 and 47.17% after 2 and 4 weeks for histomorphometry and 37.16 and 44.95% for µCT. Direct comparison showed that only the 2 weeks BA for the titanium implants was significantly different between µCT and histology (p = 0.008). Although the technique has its limitations, µCT corresponded well with histomorphometry and should be considered as a tool to evaluate bone structure around implants

Avaliação de um recobrimento de baixa espessura processado pela deposição assistida por feixe iônico como alternativa para a incorporação de biocerâmicas em implantes dentários: estudo em cães

Thin-film bioceramic coatings are potential alternatives to overcome the limitations provided by other commercially available coating techniques like PSHA, where variable bioceramic dissolution added to a metalloceramic weak link are process- inherent. The purpose of this investigation was to determine the overall and site specific (to 0.5 mm from implant surface) levels of osseoactivity around a thin-film (IBAD processed) coated titanium alloy implant versus a non surface modified (sand-blasted/acid etched) titanium alloy implant in a canine model. The surgical model comprised the proximal tibiae epiphyses with four implants placed in each limb remaining for 2 and 4 weeks in-vivo. 10 mg/Kg oxytetracycline was administered 48 hours prior to euthanization. The limbs were retrieved by sharp dissection, reduced to blocks, and subsequently nondecalcified processed for fluorescent microscopy. Micrographs (20x mag) were acquired around the implant perimeter and merged for overall biological response evaluations, and four micrographs (40x mag. subdivided in rectangles) were acquired along one of the implant sides for tetracycline labeled area fraction quantification. The results showed biocompatible and osseoconductive properties for the thin-film coated and uncoated titanium alloy implants. Tetracycline labeled area fraction analyses showed that the thin-film coated implants presented significantly higher overall and site specific osseoactivity levels at 2 and 4 weeks. The site specific osseoactivity values were significantly higher compared to overall values for control and thin-film coated implants at both times in-vivo. According to the results obtained in this study, thin-film coated implants enhanced biological response at the early implantation times evaluated.Recobrimentos biocerâmicos de baixa espessura são potenciais alternativas para compensar as limitações de outros recobrimentos biocerâmicos disponíveis comercialmente como o plasma spray de hidroxiapatita, onde a dissolução desigual e a presença de uma fraca interface metal-cerâmica são problemas inerentes ao seu processamento. O propósito desta investigação foi determinar os níveis de atividade óssea total e específica a uma área (0.5 mm da superfície do implante) ao redor de um implante de liga de titânio (superficie jateada seguida de ataque ácido) recoberto com um filme biocerâmico de baixa espessura processado através de deposição auxiliada por feixe iônico, contra um implante de liga de titânio sem recobrimento biocerâmico em cães. O modelo cirúrgico utilizou a epífise proximal da tibia, com quatro implantes colocados em cada uma, onde permaneceram por um período de 2 e 4 semanas. Oxitetraciclina (10 mg/Kg) foi administrada 48 horas antes dos animais serem sacrificados. As tíbias foram dissecadas, reduzidas a blocos, e processadas para análise em microscópio ótico. Microfotografias com aumento de 20x foram obtidas da região perimetral do implante e foram alinhadas para análise da resposta biológica total. Subsequentemente, quatro micro-fotografias com aumento de 40x, sub- divididas em retângulos, foram obtidas de um dos lados do implante para quantificação da área marcada por tetraciclina. Os resultados mostraram biocompatibilidade e osseocondutividade dos implantes de liga de titânio com ou sem filme biocerâmico de baixa espessura. Análise da área marcada por tetraciclina mostrou que os implantes com recobrimento apresentaram uma maior atividade óssea total e específica ao redor do implante em 2 e 4 semanas. Os valores de atividade óssea específica à área adjacente à superfície do implante foram significantemente maiores comparados aos valores obtidos em regiões afastadas dos implantes com ou sem recobrimento biocerâmico. De acordo com os resultados obtidos neste estudo, concluímos que os implantes com recobrimento biocerâmico de baixa espessura aumentaram a resposta biológica após 2 e 4 semanas de tempo de implantação

Podoplanin immunopositive lymphatic vessels at the implant interface in a rat model of osteoporotic fractures

Insertion of bone substitution materials accelerates healing of osteoporotic fractures. Biodegradable materials are preferred for application in osteoporotic patients to avoid a second surgery for implant replacement. Degraded implant fragments are often absorbed by macrophages that are removed from the fracture side via passage through veins or lymphatic vessels. We investigated if lymphatic vessels occur in osteoporotic bone defects and whether they are regulated by the use of different materials. To address this issue osteoporosis was induced in rats using the classical method of bilateral ovariectomy and additional calcium and vitamin deficient diet. In addition, wedge-shaped defects of 3, 4, or 5 mm were generated in the distal metaphyseal area of femur via osteotomy. The 4 mm defects were subsequently used for implantation studies where bone substitution materials of calcium phosphate cement, composites of collagen and silica, and iron foams with interconnecting pores were inserted. Different materials were partly additionally functionalized by strontium or bisphosphonate whose positive effects in osteoporosis treatment are well known. The lymphatic vessels were identified by immunohistochemistry using an antibody against podoplanin. Podoplanin immunopositive lymphatic vessels were detected in the granulation tissue filling the fracture gap, surrounding the implant and growing into the iron foam through its interconnected pores. Significant more lymphatic capillaries were counted at the implant interface of composite, strontium and bisphosphonate functionalized iron foam. A significant increase was also observed in the number of lymphatics situated in the pores of strontium coated iron foam. In conclusion, our results indicate the occurrence of lymphatic vessels in osteoporotic bone. Our results show that lymphatic vessels are localized at the implant interface and in the fracture gap where they might be involved in the removal of lymphocytes, macrophages, debris and the implants degradation products. Therefore the lymphatic vessels are involved in implant integration and fracture healing

Proteolytic and Mechanical Matrix Remodeling During Capillary Morphogenesis

Engineering large viable tissues requires techniques for encouraging rapid capillary bed formation to prevent necrosis. A convenient means of creating this micro-vascular network is through spontaneous neovascularization, which occurs when endothelial cells (ECs) and supportive stromal cells are co-encapsulated within a variety of hydrogel-based extracellular matrices (ECM) and self-assemble into an interconnected network of endothelial tubules. Although this is a robust phenomenon, the environmental and cell-specific determinants that affect the rate and quality of micro-vascular network formation still require additional characterization to improve clinical translatability. This thesis investigates how the proteolytic susceptibility of engineered matrices effects neovascular self-assembly in poly(ethylene glycol) (PEG) hydrogels and provides characterization of changes to matrix mechanics that accompany neovascular morphogenesis in fibrin and PEG hydrogels. Proteolytic ECM remodeling is essential for the process of capillary morphogenesis. Pharmacological inhibitor studies suggested a role for both matrix metalloproteinases (MMP)- and plasmin-mediated mechanisms of ECM remodeling in an EC-fibroblast co-culture model of vasculogenesis in fibrin. To further investigate the potential contribution of plasmin mediated matrix degradation in facilitating capillary morphogenesis we employed PEG hydrogels engineered with proteolytic specificity to either MMPs, plasmin, or both. Although fibroblasts spread in plasmin-selective hydrogels, we only observed robust capillary morphogenesis in MMP-sensitive matrices, with no added benefit in dual susceptible hydrogels. Enhanced capillary morphogenesis was observed, however, in PEG hydrogels engineered with increased susceptibility to MMPs without altering proteolytic selectivity or hydrogel mechanical properties. These findings highlight the critical importance of MMP-mediated ECM degradation during vasculogenesis and justify the preferential selection of MMP-degradable peptide crosslinkers in the design of synthetic hydrogels used to promote vascularization. Matrix stiffness is a well-established cue in cellular morphogenesis, however, the converse effect of cellular remodeling on environmental mechanics is comparatively under characterized. In fibrin hydrogels, we applied traditional bulk rheology and laser tweezers-based active microrheology to demonstrate that both ECs and fibroblasts progressively stiffen the ECM across length scales, with the changes in bulk properties dominated by fibroblasts. Despite a lack of fibrillar architecture, a similar stiffening effect was observed in MMP-degradable PEG hydrogels. This stiffening tightly correlated with degree of vessel formation and critically depended on active cellular contractility. To a lesser degree, deposition of ECM proteins also appeared to contribute to progressive hydrogel stiffening. Blocking cell-mediated hydrogel degradation abolished stiffening, demonstrating that matrix metalloproteinase (MMP)-mediated remodeling is required for stiffening to occur. EC co-culture with mesenchymal stem cells (MSCs) in PEG resulted in reduced vessel formation compared to fibroblast co-cultures and no change in hydrogel mechanics over time. The correlation between matrix stiffening and enhanced vessel formation, and dependence on cellular contractility, suggests differences in vessel formation between fibroblasts and MSCs may be partially mediated by differences in cellular contractility. Collectively, these findings provide a deeper understanding of mechanobiological effects during capillary morphogenesis and highlight the dynamic reciprocity between cells and their mechanical environmentPHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/162935/2/bjuliar_1.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/162935/1/bjuliar_2.pd

Perfusion bioreactor culture of human adipose-derived stromal cells on decellularized adipose tissue scaffolds enhances in vivo adipose tissue regeneration

Tissue-engineering approaches hold promise to address the need in plastic and reconstructive surgery for new therapies that promote stable adipose tissue regeneration. Previous studies have demonstrated the potential of combining decellularized adipose tissue (DAT) scaffolds with adipose-derived stromal cells (ASCs) for volume augmentation applications. With the goal of enhancing in vivo angiogenesis and adipogenesis, this study evaluated the effects of culturing human ASCs on DAT scaffolds within a perfusion bioreactor. Using this system, the impact of both dynamic culture and hypoxic preconditioning were explored in vitro and in vivo. Initial studies compared the effects of 14 days of culture within the perfusion bioreactor under hypoxia (2% O2) or normoxia (~20% O2) on human ASC expansion and expression of hypoxia inducible factor-1 alpha (HIF-1α) in vitro relative to static cultured controls. The findings indicated that culturing within the bioreactor under hypoxia significantly increased ASC proliferation on the DAT, with a higher cell density observed in the scaffold periphery. Subsequent characterization in a subcutaneous implant model in athymic nude mice revealed that in vivo angiogenesis and adipogenesis were markedly enhanced when the ASCs were cultured on the DAT within the perfusion bioreactor under hypoxia for 14 days prior to implantation relative to the other culture conditions, as well as freshly seeded and unseeded DAT control groups. Overall, dynamic culture within the perfusion bioreactor system under hypoxia represents a promising approach for preconditioning ASCs on DAT scaffolds to enhance their capacity to stimulate angiogenesis and host-derived adipose tissue regeneration

The Application of Melatonin and Platelet-Rich Plasma in the Development of a Bioactive Calcium Aluminate Bone Regenerative Scaffold

Over 500,000 bone graft procedures are conducted annually within the United States. Autografts contribute to donor site complications and disease transmission with allografts has been described. Many ceramics are only osteoconductive and are brittle, limiting their clinical use. Thus, the objective of this study was to create a bone substitute with osteoinductive properties similar to natural bone using the ceramic biomaterial calcium aluminate (CA). Calcium aluminate materials are durable and remain moldable for an extended period of time at room temperature. Further, the surfaces of CA scaffolds can be modified with biological agents through simple chemical means to locally deliver agents directly to sites of injury. In order to enhance local bone regenerating characteristics of CA scaffolds, melatonin and platelet-rich plasma (PRP) were utilized for their known osteoinductive properties. Platelet-rich plasma enhances soft and hard tissue formation primarily through growth factor-mediated signaling pathways. Melatonin augments osteoblast differentiation and inhibits osteoclast-mediated bone resorption through receptor-dependent signaling and free radical scavenging activity, respectively. Thus, it was hypothesized that melatonin and/or PRP would provide osteoinductive properties to CA scaffolds to promote bone regeneration in a rodent model of critical-size calvaria defects. Modified CA scaffolds (CA-Mel) were produced by immobilizing melatonin to the CA surface through a covalent linkage. The biocompatibility of unmodified and modified CA scaffolds was initially tested in vitro and indicated that modified surfaces had a preference for the adhesion and proliferation of normal human osteoblasts versus NIH 3T3 fibroblasts. Moreover, the immobilization of melatonin to the CA surface may delay the differentiation of human adult mesenchymal stem cells (hAMSCs) and may have facilitated their migration across the CA surface. Two-month-old ovariectomized rats were randomized into implant groups receiving unmodified or modified scaffolds in the absence (CA and CA-Mel) or presence of PRP (CA+PRP and CA-Mel+PRP). Histological sections confirmed that both CA scaffold types were well-tolerated and provided evidence of tissue infiltration and scaffold biodegradation over time. Bone regeneration in animals was assessed by fluorochrome labeling at three and six months. While there was a lack of synergism between melatonin and PRP in the CA-Mel+PRP group, animals implanted with CA-Mel showed the greatest intensity and abundance of bone remodeling at both time points compared to all other groups. Radiographic data indicated a significant increase in the density of newly formed bone over time in all groups. The absence of a detectable decrease in density suggests that the modest biodegradation of CA scaffolds is balanced with processes of bone formation. Finally, both unmodified and modified CA scaffolds continued to provide a supportive surface for bone formation out to six months. Overall, results from this study suggest that CA scaffolds modified with melatonin may enhance bone remodeling activity in calvarial defects through hAMSC differentiation and recruitment and by preferentially supporting the viability and function of mature osteoblasts. This novel bioactive ceramic scaffold has the potential to change the dogma of bone grafting in fields like dentistry and reconstructive surgery. Continued optimization of this therapy is warranted and the attachment of other osteoinductive biomolecules is being considered

Immediate versus delayed loading: comparison of primary stability loss after miniscrew placement in orthodontic patients-a single-centre blinded randomized clinical trial

Introduction: The aim of this randomized clinical trial was to compare torque recordings at insertion time and 1 week post-placement between immediately loaded orthodontic miniscrews and an unloaded control group. Trial design: This RCT was designed as parallel with an allocation ratio of 1:1. Methods: Eligibility criteria to enroll patients were: needs of fixed orthodontic treatment, no systemic disease, absence of using drugs altering bone metabolism. All patients were consecutively treated in a private practice and the miniscrews were placed by the same author. Patients received ORTHOImplant (3M Unitek) miniscrews and they were blindly divided in two groups: group 1 screws were unloaded between T0 and T1, group 2 received immediately loaded screws with NiTi coil. For each patient, maximum insertion torque (MIT) was evaluated at T0. After 1 week, without loading, the screw torque was measured again (T1) and at the end of the treatment maximal removal torque was evaluated (T2). Torque variation in the first week was considered as the primary outcome. Randomization: A randomization list was created for the group assignment, with an allocation ratio of 1:1. Blinding: The study was single blinded in regard of the statistical analysis. Results: Patients enrolled in the clinical trial were 51 for a total of 81 miniscrews. The recruitment started in November 2012 and the observation period ended in August 2014. Twenty-six and twenty-five patients were analysed in group 1 and 2, respectively. The MIT mean in each placement time was 18.25 Ncm (SD = 3.00), 11.41 Ncm (SD = 3.51) and 10.52 Ncm (SD = 5.14) at T0, T1, and T2 time, respectively. In group 1, the torque decrease between T1 and T0 was statistically higher compared to group 2 (P value = 0.003). Statistically significant effects of the placement times on MIT were found (P value <0.0001). No serious harm was observed. Limitations: This study was performed using only direct force on the miniscrew and not using the miniscrew as an indirect anchorage. It was not possible to obtain quantitative data on bone quality or root proximity to miniscrews. Conclusions: A significant stability loss was observed in the first week in both groups; Group 1 showed a statistically higher torque loss in the first week when compared to the immediately loaded group. There were statistically significant effects of the measurement times on MIT and of the miniscrew location on MIT. The overall failure rate was 7.4%. Trial registration: This trial was not registered. Protocol: The protocol was not published before trial commencement

Osseointegrated Oral implants

In the past, osseointegration was regarded to be a mode of implant anchorage that simulated a simple wound healing phenomenon. Today, we have evidence that osseointegration is, in fact, a foreign body reaction that involves an immunologically derived bony demarcation of an implant to shield it off from the tissues. Marginal bone resorption around an oral implant cannot be properly understood without realizing the foreign body nature of the implant itself. Whereas the immunological response as such is positive for implant longevity, adverse immunological reactions may cause marginal bone loss in combination with combined factors. Combined factors include the hardware, clinical handling as well as patient characteristics that, even if each one of these factors only produce subliminal trauma, when acting together they may result in loss of marginal bone. The role of bacteria in the process of marginal bone loss is smaller than previously believed due to combined defense mechanisms of inflammation and immunological reactions, but if the defense is failing we may see bacterially induced marginal bone loss as well. However, problems with loss of marginal bone threatening implant survival remains relatively uncommon; we have today 10 years of clinical documentation of five different types of implant displaying a failure rate in the range of only 1 to 4 %

Vascular Endothelial Growth Factor (VEGF) and Semaphorin 3A (Sema3A) signaling for vascularized bone grafts

One of the major challenges for the treatment of critical size bone defects is to ensure a rapid and efficient vascularization of tissue-engineered bone grafts upon implantation in vivo. The biological processes of osteogenesis and angiogenesis are intimately coupled, and many factors play important roles in this cross-talk. Among them, Vascular Endothelial Growth Factor (VEGF), the master regulator of vascular growth, is crucial during bone development, homeostasis and repair, and it is a key molecular target for the generation of vascularized bone grafts. However, in order to exploit its therapeutic potential, VEGF dose and spatial-temporal distribution have to be precisely controlled. Semaphorin 3A (Sema3A) regulates osteoblasts and osteoclasts to promote bone synthesis through the Neuropilin-1 receptor (NP-1) and it has important roles in angiogenesis. We previously found that VEGF dose-dependently inhibits endothelial Sema3A expression in skeletal muscle. Here we investigated the role of VEGF and Sema3A in the coupling of angiogenesis and osteogenesis in engineered bone grafts in order to provide rational bases for novel, safe and effective therapeutic strategies for the repair of bone tissue To this purpose, osteogenic constructs were prepared with human bone marrow mesenchymal cells (BMSCs) in combination with fibrin matrices decorated with recombinant VEGF or Sema3A engineered with a transglutaminase substrate sequence (TG-VEGF and TG-Sema3A) to allow cross-linking into fibrin hydrogels and controlled release. We found that VEGF-dose dependently regulates both angiogenesis and osteogenesis. Low VEGF doses accelerated vascular invasion and ensured efficient bone depositio. High VEGF doses delayed vascular ingrowth, increased osteoclast recruitment and decreased bone formation by impairing the differentiation of the implanted human osteogenic progenitor cells. Moreover, we showed that VEGF-dose dependently downregulates Sema3A expression and that Sema3A is critical for both vascularization and intramembranous bone formation in osteogenic grafts. These results confirm the importance of both VEGF and Sema3A in bone biology and provide the basis for the design of novel rational strategies to generate vascularized bone grafts with the aim to improve the healing of critical-size bone defects