Marine Collagen/Apatite scaffolds envisaging tissue engineering applications

Despite the vast investigation and the large amount of products already available in the market to treat the different bone defects there is still a growing need to develop more advanced and complex therapeutic strategies. In this context, a mixture of Marine Hydroxyapatite-Fluorapatite:Collagen (HA-FP:ASC) seems to be a promising solution to overcome these bone defects, specifically, dental defects. HA-FP particles (20–63 μm) were obtained through pyrolysis (950°C, 12 h) of shark teeth (Isurus oxyrinchus, P. glauca), and Type I collagen was isolated from Prionace glauca skin as previously described (1). After the steps of purification, collagen was solubilized in 0.5 M acetic acid and HA-FP added producing three different formulations: were produced, 30:70, 50:50 and 70:30 of HA-FP:ASC, respectively. EDC/NHS and HMDI binding agents were used to stabilize the produced scaffolds. Mechanical properties were evaluated by compression tests. SEM analysis allowed observing the mineral deposition, after immersion in simulated body fluid and also permitted to evaluate how homogenous was the distribution of HA-FP in the different scaffold formulations, also confirmed by μ-CT assay. It was readily visible by Cytotoxicity and life/dead CLSM assays that cells were able to adhere and proliferate in the produced scaffolds. Scaffolds crosslinked with EDC/NHS showed lower cytotoxicity, being the ones chosen for further cellular evaluation.

Evaluación de la regeneración ósea mediante procesado de imagen

La regeneració òssia és un procés estudiat per experts de tot el món. Aquests experts estudien materials capaços d’accelerar el procés de formació de teixit ossi en zones on s’han produït defectes ossis. Després d’un determinat període de temps de l’aplicació dels materials d’estudi en la zona on hi havia una manca de teixit ossi, s’obtenen imatges d’aquesta zona on l’expert mitjançant l’ inspecció visual d’aquestes imatges avalua si l’os s’ha regenerat bé o no. El problema d’aquest mètode d’avaluació es que requereix d’un expert on la valoració d’aquest és subjectiva i difícil de quantificar, el que pot provocar que hi hagi discordança entre experts. Amb la finalitat de aprofitar les imatges en que es basa l’expert per avaluar la capacitat de regeneració òssia dels materials d’estudi es proposa realitzar un anàlisi quantitatiu de la regeneració òssia basat en el processament d’imatge. L’algorisme dissenyat es capaç de classificar imatges de la mandíbula en: imatges de regeneració bona i dolenta mitjançant la parametrització de l’histograma de nivells de grisos de la imatge, solucionant la falta d’objectivitat del mètode d’avaluació de la regeneració òssia i la necessitat d’un expert per realitzar-la.

Prefabrication of composite grafts for long-segment tracheal reconstruction.

OBJECTIVE: To investigate the prefabrication of vascularized mucosa-lined composite grafts intended to replace circumferential tracheal defects. DESIGN: Plane grafts composed of ear cartilage and full-thickness oral mucosa were revascularized by the laterothoracic fascia. The use of meshed vs nonmeshed mucosa to improve the epithelial coverage was examined. We also investigated the creation of a vascular bed over the cartilage and the subsequent application of meshed mucosa. Macroscopic aspects, viability, and degree of mucosal lining were analyzed. SUBJECTS: Twenty male New Zealand white rabbits. INTERVENTIONS: Ten animals underwent placement of auricular cartilage under the laterothoracic fascia. Intact (group 1) or meshed mucosa (group 2) was applied over the fascia and protected by a silicone sheet. After 3 weeks, prefabricated grafts were removed for comparison. In 10 other animals, a sheet of perforated cartilage was placed under the laterothoracic fascia. Two weeks later, 5 grafts (group 3) were harvested. The remaining 5 grafts were reopened for mucosal application over the cartilage and revascularized for 3 additional weeks (group 4). RESULTS: Vascularized plane grafts were obtained in all groups. Mucosal lining increased significantly with meshed mucosa (14%-68%; mean, 40%) compared with nonmeshed mucosa (3%-15%; mean, 10%) (P = .008). Induction of a vascular bed over perforated cartilage was achieved, but survival of secondary implanted mucosa was variable. CONCLUSIONS: A reliable technique to prefabricate composite grafts with cartilaginous support and mucosal lining is presented. The use of meshed mucosa significantly improves epithelial coverage.

Thermoplastic bioactive composite – with special reference to dissolution behaviour and tissue response

Bioactive glasses are surface-active ceramic materials which support and accelerate bone growth in the body. During the healing of a bone fracture or a large bone defect, fixation is often needed. The aim of this thesis was to determine the dissolution behaviour and biocompatibility of a composite consisting of poly(ε-caprolactone-co-DL-lactide) and bioactive glass (S53P4). In addition the applicability as an injectable material straight to a bone defect was assessed. In in vitro tests the dissolution behaviour of plain copolymer and composites containing bioactive glass granules was evaluated, as well as surface reactivity and the material’s capability to form apatite in simulated body fluid (SBF). The human fibroblast proliferation was tested on materials in cell culture. In in vivo experiments, toxicological tests, material degradation and tissue reactions were tested both in subcutaneous space and in experimental bone defects. The composites containing bioactive glass formed a unified layer of apatite on their surface in SBF. The size and amount of glass granules affected the degradation of polymer matrix, as well the material’s surface reactivity. In cell culture on the test materials the human gingival fibroblasts proliferated and matured faster compared with control materials. In in vitro tests a connective tissue capsule was formed around the specimens, and became thinner in the course of time. Foreign body cell reactions in toxicological tests were mild. In experimental bone defects the specimens with a high concentration of small bioactive glass granules (<45 μm) formed a dense apatite surface layer that restricted the bone ingrowth to material. The range of large glass granules (90-315 μm) with high concentrations formed the best bonding with bone, but slow degradation on the copolymer restricted the bone growth only in the superficial layers. In these studies, the handling properties of the material proved to be good and tissue reactions were mild. The reactivity of bioactive glass was retained inside the copolymer matrix, thus enabling bone conductivity with composites. However, the copolymer was noticed to degradate too slowly compared with the bone healing. Therefore, the porosity of the material should be increased in order to improve tissue healing.

Résultats des cupules de grand diamètre dans les reprises d’arthroplastie totale de hanche : suivi minimum de deux ans.

La révision d’arthroplastie de la hanche en cas d’important déficit osseux acétabulaire peut être difficile. Les reconstructions avec cupule de très grand diamètre ou cupule « jumbo » (≥ 62 mm chez la femme et ≥ 66 mm chez l’homme) sont une option thérapeutique. Nous voulions évaluer la préservation et la restauration du centre de rotation de la hanche reconstruite en la comparant au coté controlatéral sain ou selon les critères de Pierchon et al. Nous voulions également évaluer la stabilité du montage à un suivi d’au moins 2 ans. Il s’agissait de 53 cas consécutifs de révision acétabulaire pour descellement non septique avec implantation d’une cupule jumbo sans ciment à l’Hôpital Maisonneuve-Rosemont. Le déficit osseux évalué selon la classification de Paprosky et al. Les cupules implantées avaient un diamètre moyen de 66 mm (62-81) chez les femmes et 68 mm (66-75) chez les hommes. L’allogreffe osseuse morcelée et massive était utilisée dans 34 et dans 14 cas respectivement. La cupule a été positionnée avec un angle d’inclinaison moyen de 41.3° (26.0-53.0). Le centre de rotation de la hanche reconstruite a été jugé satisfaisant dans 78% de cas sur l'axe médiolatéral, 71% sur l'axe craniopodal et amélioré dans 27% dans cet axe. Au recul moyen radiologique de 84.0 mois (24.0-236.4) et clinique de 91.8 mois (24.0 – 241.8): 6 cas étaient décédés, 3 perdus au suivi. On a observé le descellement radiologique dans un 1 cas, la luxation récidivante dans 5 cas et l’infection dans 4 cas. Le retrait de la cupule a été effectué dans 2 cas pour infection. L’ostéointégration des greffons osseux était complète dans tous les cas sauf 3. Les scores cliniques étaient pour le HHS de 82 +/-17, le WOMAC de 86 +/- 14 et le SF-12 physique de 46 +/- 12 et mental 53 +/-13. La cupule jumbo peut être considérée comme un moyen fiable pour gérer le déficit osseux dans les révisions acétabulaires. Elle permet de conserver ou d’améliorer la position du centre de rotation physiologique de la hanche. La fixation sans ciment favorise l’ostéointégration de la cupule et permet une stabilité à moyen terme. Le taux de complications est comparable ou inférieur à d'autres procédures de reconstruction.

Avaliação microscópica dos fragmentos ósseos obtidos por diferentes métodos de osteotomia e de irrigação em aloenxertos irradiados e congelados de coelho

Oral and facial bone defects can undertake appearance, psychosocial well-being and stomathognatic function of its patients. Over the yerars several strategies for bone defect regeneration have arised to treat these pathologies, among them the use of frozen and irradiated bone allograft. Manipulation of bone grafts it s not determined yet, and several osteotomy alternatives can be observed. The present work evaluated with a microscope the bone fragments obtained from different osteotomy methods and irrigation on rings and blocks allografts irradiated and frozen at 80° negative in a rabbit model. The study is experimental in vitro and it sample was an adult male New Zealand rabbit. The animal was sacrificed to obtain long bones, that were submitted to freezing at 80º negative and irradiated with Cobalt- 60. Then the long bones were sectioned into 24 bone pieces, divided into 4 groups: G1 (n=06) osteotomy was performed with bur No. 6 forming rings with 5 mm thickness with high-speed handpiece with manual irrigation; G2 (n=06) osteotomy was performed with bur No. 6 forming rings with 5 mm thick with surgical motor with a manual irrigation rotation 1500 rpm; GA (n=06), osteotomy with trephine using manual irrigation with saline; and GB (n=06), osteotomy with trephine using saline from peristaltic pumps of surgical motor. Five bone pieces of each group were prepared for analysis on light microscopy (LM) and one on electronic scan electronic microscopy (SEM). On the SEM analysis edges surface, presence of microcracks and Smear Layer were evaluated. Analyzing osteotomy technics on SEM was observed: increased presence of microcracks cutting with high speed; increased presence of areas covered by Smear Layer when cutting with motor implant. The irrigation analysis with SEM was observed: that the presence of microcracks does not depend on the type of irrigation; on manual irrigation, there was greater discrepancy between the cutting lines. The descriptive analysis of the osteotomy and irrigation process on LM showed: histological analysis showing the bony margins with clear tissue changed layer, composed of blackened tissue of charred appearance near to the cortical bone; on the edges of the bony part, bone fragments that were displaced during the bone cut and bone irregularities were observed. After analysis of results we can conclude: that there was greater regularity of the bone cut using high-speed handpiece than using motor implant; the cut with trephine using saline irrigated from peristaltic pumps of surgical motor showed greater homogeneity when compared with manual irrigation; charred tissue was found in all obtained bone samples, whit no significant statistically difference on the proportion of carbonization of the two analysed technics

Estudo da descelularização tecidual na produção de arcabouços biológicos para enxertos

The regeneration of bone defects with loss of substance remains as a therapeutic challenge in the medical field. There are basically four types of grafts: autologous, allogenic, xenogenic and isogenic. It is a consensus that autologous bone is the most suitable material for this purpose, but there are limitations to its use, especially the insufficient amount in the donor. Surveys show that the components of the extracellular matrix (ECM) are generally conserved between different species and are well tolerated even in xenogenic recipient. Thus, several studies have been conducted in the search for a replacement for autogenous bone scaffold using the technique of decellularization. To obtain these scaffolds, tissue must undergo a process of cell removal that causes minimal adverse effects on the composition, biological activity and mechanical integrity of the remaining extracellular matrix. There is not, however, a conformity among researchers about the best protocol for decellularization, since each of these treatments interfere differently in biochemical composition, ultrastructure and mechanical properties of the extracellular matrix, affecting the type of immune response to the material. Further down the arsenal of research involving decellularization bone tissue represents another obstacle to the arrival of a consensus protocol. The present study aimed to evaluate the influence of decellularization methods in the production of biological scaffolds from skeletal organs of mice, for their use for grafting. This was a laboratory study, sequenced in two distinct stages. In the first phase 12 mice hemi-calvariae were evaluated, divided into three groups (n = 4) and submitted to three different decellularization protocols (SDS [group I], trypsin [Group II], Triton X-100 [Group III]). We tried to identify the one that promotes most efficient cell removal, simultaneously to the best structural preservation of the bone extracellular matrix. Therefore, we performed quantitative analysis of the number of remaining cells and descriptive analysis of the scaffolds, made possible by microscopy. In the second stage, a study was conducted to evaluate the in vitro adhesion of mice bone marrow mesenchymal cells, cultured on these scaffolds, previously decellularized. Through manual counting of cells on scaffolds there was a complete cell removal in Group II, Group I showed a practically complete cell removal, and Group III displayed cell remains. The findings allowed us to observe a significant difference only between Groups II and III (p = 0.042). Better maintenance of the collagen structure was obtained with Triton X-100, whereas the decellularization with Trypsin was responsible for the major structural changes in the scaffolds. After culture, the adhesion of mesenchymal cells was only observed in specimens deccelularized with Trypsin. Due to the potential for total removal of cells and the ability to allow adherence of these, the protocol based on the use of Trypsin (Group II) was considered the most suitable for use in future experiments involving bone grafting decellularized scaffolds

Mistura de proteínas morfogenéticas ósseas, hidroxiapatita, osso inorgânico e colágeno envolta por membrana de pericárdio no preenchimento de defeito ósseo segmentar em coelhos

Avaliou-se o uso de biomaterial de origem bovina na regeneração de defeitos ósseos segmentares empregando-se 12 coelhos, fêmeas, da raça Norfolk, com idade de seis meses e pesos entre 3 e 4,5kg. Realizou-se falha segmentar bilateral de um centímetro de comprimento na diáfise do rádio, com inclusão do periósteo. No membro direito, o defeito foi delimitado por membrana de pericárdio liofilizada, contendo em seu interior mistura de proteínas morfogenéticas ósseas adsorvidas a hidroxiapatita, colágeno liofilizado e osso inorgânico. No membro esquerdo, o defeito não recebeu tratamento. Radiografias foram obtidas ao término do procedimento cirúrgico e aos sete, 30, 60, 90, 120 e 150 dias de pós-operatório. Após eutanásia de seis coelhos aos 60 dias e seis aos 150 dias de pós-cirúrgico, os resultados radiográficos e histológicos mostraram que a regeneração óssea foi inibida nos defeitos segmentares tratados com o biomaterial.

Autogenous demineralized dentin matrix for tissue engineering applications: Radiographic and histomorphometric studies

Purpose: This work evaluated the osteoconductive properties of autogenous demineralized dentin matrix (ADDM) on surgical bone defects in the parietal bone of rabbits, using the guided bone regeneration technique and polytetrafluoroethylene (PTFE) membrane. Materials and Methods: Surgical bone defects were created in 24 adult rabbits and repaired with either ADDM and PTFE (experimental group) or PTFE alone (control group). The ADDM had been obtained from the central incisors of the experimental rabbits. The rabbits were sacrificed after 15, 30, 60, and 90 days and the defects examined radiographically and histologically. Results: Radiographically, the defects in the experimental animals achieved radiopacity more quickly than the defects in the control group. Discussion: After 15, 30, 60, and 90 days of observation following surgery, the ADDM slices appeared to stimulate new bone formation. The dentin slices were completely incorporated into the new bone tissue and were resorbed during the bone repair. Conclusions: Bone repair was accelerated on the bone defects treated with ADDM when compared to the control group.

Healing outcomes at implants installed in grafted sites: an experimental study in dogs

Aim: To evaluate the integration of implants installed using a surgical guide in augmented sites with autologous bone or deproteinized bovine bone mineral (DBBM) blocks, concomitantly with a collagen membrane.Material and methods: Mandibular molars were extracted bilaterally in six Labrador dogs, the buccal bony wall was removed, and a box-shaped defect was created. After 3 months, flaps were elevated, a bony graft was harvested from the ascending ramus, and secured to the lateral wall of the defect by means of screws. In the left mandibular side, a DBBM block was fixed into the defect. A resorbable membrane was applied at both sides, and the flaps were sutured. After 3 months, flaps were elevated, and a customized device was used as surgical guide to prepare the recipient sites in the interface between grafts and parent bone. One implant was installed in each side of the mandible. After 3 months, biopsies were harvested, and ground sections were prepared for histologic evaluation.Results: One autologous bone block graft was lost before implant installation. The width of the alveolar crest at the test sites (DBBM) was 5.4 +/- 1.2 mm before, 9.4 +/- 1.2 mm immediately after grafting, and 9.3 +/- 1 mm at implant installation. At the control sites (autologous bone), the corresponding values were: 5.2 +/- 1, 9 +/- 1.2, and 8.7 +/- 0.9 mm, respectively. All implants installed were available for histologic evaluation (n = 5). The autologous bone grafts, rich in vessels and cells, were integrated in the parent bone, and only little non-vital bone was found. The BIC% was 56.7 +/- 15.6% and 54.2 +/- 13.2% at the buccal and lingual aspects, respectively. At the test sites, the DBBM appeared to be embedded into connective tissue, and very little newly formed bone was encountered within the grafts. The BIC% was 5.8 +/- 12.3% and 51.3 +/- 14.2% at the buccal and lingual aspects, respectively.Conclusions: Autologous bone blocks used to augment the alveolar bony crest horizontally allowed the complete osseointegration of implants installed after 3 months of healing. However, similar blocks of DBBM did not promote osseointegration, although the installed implants were stable owing to the osseointegration in the sites of the parent bone.

Histomorphometric analysis of homogenous demineralized dentin matrix as osteopromotive material in rabbit mandibles

Purpose: The aim of this work was to evaluate the effectiveness of homogenous demineralized dentin matrix (HDDM) slices in surgical bone defects created in the mandibles of rabbits and occluded with a polytetrafluoroethylene (PTFE) membrane in the promotion of bone growth. Materials and Methods: Surgical bone defects were created in 36 adult rabbits and divided into 4 groups: bone defect (control), bone defect with PTFE membrane, bone defect with HDDM, and bone defect with both HDDM and a PTFE membrane (HDDM + PTFE). The rabbits were sacrificed after 30, 60, and 90 days, and the bone defects were examined histologically and by histomorphometric analysis (analysis of variance and the Tukey test). Results: The volume of newly formed bone matrix was significantly greater in the HDDM and HDDM + PTFE groups than in the control and PTFE groups. The discrete inflammatory reaction found in the HDDM and HDDM + PTFE groups did not prevent the osteopromotive activity of the dentin matrix. Discussion: HDDM slices were biocompatible and were resorbed during the bone remodeling process. They stimulated the newly formed bone until 30 days after implantation. Conclusion: Bone repair was accelerated in the bone defects treated with HDDM in comparison to the control group.

Porous polyethylene for tissue engineering applications in diabetic rats treated with calcitonin: Histomorphometric analysis

Purpose: The purpose of this work was to study the bone tissue reaction after porous polyethylene (Polipore) implantation into surgical defects in the parietal bones of rats with streptozotocin-induced diabetes, treated with salmon calcitonin. Materials and Methods: Porous polyethylene implants were placed in bone defects created in 36 adult female rats. The rats were divided into 3 equal groups: diabetic treated with calcitonin (DCa), diabetic (D), and control (C). The animals of the DCa group received applications of salmon calcitonin on alternating days immediately after the surgery until sacrifice. The rats were sacrificed after 15, 30, 60, and 90 days, and the defects were examined histologically and statistically through histomorphometric analysis. Results: Histomorphometric analysis showed that there was no statistically significant difference in the mean quantity of inflammatory cells among all study groups after 15 and 90 days. At 30 days, a statistically significant difference was observed between the D and C groups and the D and DCa groups. At 60 days, there was no statistically significant difference between the D and DCa groups. Discussion: Porous polyethylene can be considered an option for implant material when there are investigations that prove its biocompatibility and stability in the host tissues. Salmon calcitonin positively aided the bone repair and attenuated the inflammatory response until 30 days after the surgery. Conclusion: Porous polyethylene was tolerated by the host tissues in all groups, and moderate chronic inflammatory reaction was observed up to the 90-day period. Salmon calcitonin attenuated the inflammatory response up until 30 days.

Failure of miniplate osteosynthesis for the management of atrophic mandibular fracture

Fractures of the severely atrophic (<10 mm) edentulous mandible are not common, and these fractures with a vertical height of 10 mm or less have long been recognized as being particularly problematic. Although there are advances in the treatment of the atrophic mandibular fracture, the treatment remains controversial. There are some options for treatment planning because of using small miniplates to large reconstruction plates. However, when the fixation method fails, it causes malunion, nonunion, and/or infection, and sometimes it has been associated with large bone defects. The authors describe a clinical report of a failed miniplate fixation for atrophic mandibular fracture management. The authors used a load-bearing reconstruction plate combined with autogenous bone graft from iliac crest for this retreatment. The authors show a follow-up of 6 months, with union of the fracture line and no complication postoperatively. © 2013 by Mutaz B. Habal, MD.

Reparação óssea de cavidades cirurgicamente criadas e preenchidas com uma associação de vidro bioativo e osso autógeno: análise histométrica e imunoistoquímica

Pós-graduação em Odontologia - FOA

Avaliação dos enxertos ósseos autógenos triturados manualmente ou coletados durante osteotomia: análise histológica e histométrica em coelhos

Pós-graduação em Odontologia - FOA