Marcelo Rodrigues da Cunha

A new heterologous fibrin sealant as scaffold to recombinant human bone morphogenetic protein-2 (rhBMP-2) and natural latex proteins for the repair of tibial bone defects.

Tissue engineering has special interest in bone tissue aiming at future medical applications Studies have focused on recombinant human bone morphogenetic protein-2 (rhBMP-2) and natural latex proteins due to the osteogenic properties of rhBMP-2 and the angiogenic characteristic of fraction 1 protein (P-1) extracted from the rubber tree Hevea brasiliensis. Furthermore, heterologous fibrin sealant (FS) has been shown as a promising alternative in regenerative therapies. The aim of this study was to evaluate these substances for the repair of bone defects in rats. A bone defect measuring 3mm in diameter was created in the proximal metaphysis of the left tibia of 60 rats and was implanted with rhBMP-2 or P-1 in combination with a new heterologous FS derived from snake venom. The animals were divided into six groups: control (unfilled bone defect), rhBMP-2 (defect filled with 5μg rhBMP-2), P-1 (defect filled with 5μg P-1), FS (defect filled with 8μg FS), FS/rhBMP-2 (defect filled with 8μg FS and 5μg rhBMP-2), FS/P-1 (defect filled with 8μg FS and 5μg P-1). The animals were sacrificed 2 and 6 weeks after surgery. The newly formed bone projected from the margins of the original bone and exhibited trabecular morphology and a disorganized arrangement of osteocyte lacunae. Immunohistochemical analysis showed intense expression of osteocalcin in all groups. Histometric analysis revealed a significant difference in all groups after 2 weeks (p<0.05), except for the rhBMP-2 and FS/rhBMP-2 groups (p>0.05). A statistically significant difference (p<0.05) was observed in all groups after 6 weeks in relation to the volume of newly formed bone in the surgical area. In conclusion, the new heterologous fibrin sealant was found to be biocompatible and the combination with rhBMP-2 showed the highest osteogenic and osteoconductive capacity for bone healing. These findings suggest a promising application of this combination in the regeneration surgery.

Effects of chronic passive smoking on the regeneration of rat femoral defects filled with hydroxyapatite and stimulated by laser therapy

Defects associated with bone mass loss are frequently treated by autogenous bone grafting. However, synthetic biomaterials such as calcium phosphate ceramics can substitute autologous grafts as long as they are biocompatible with bone tissue. In addition, low-level laser therapy (LLLT) is used to enhance bone regeneration by stimulating the local microcirculation and increasing the synthesis of collagen by bone cells. However, bone health is fundamental for osseointegration of the graft and bone repair. In this respect, excessive tobacco consumption can compromise expected outcomes because of its deleterious effects on bone metabolism that predispose to the development of osteoporosis. The objective of this study was to evaluate the regeneration of bone defects implanted with biomaterial and stimulated by LLLT in rats submitted to passive cigarette smoking. Porous hydroxyapatite granules were implanted into critical-size defects induced experimentally in the distal epiphysis of the right femur of 20 female Wistar rats submitted to passive smoking for 8 months in a smoking box. The defect site was irradiated with a gallium-arsenide laser at an intensity of 5.0 J/cm2. The animals were divided into four groups: control (non-smoking) rates submitted (G2) or not (G1) to laser irradiation, and smoking rats submitted (G4) or not (G3) to laser irradiation. The animals were sacrificed 8 weeks after biomaterial implantation. The right femurs were removed for photodocumentation, radiographed, and processed for routine histology. The results showed good radiopacity of the implant site and of the hydroxyapatite granules. Histologically, formation of new trabecular bone was observed adjacent to the hydroxyapatite granules in G1 and G2. In G3 and G4, the granules were surrounded mainly by connective tissue. In conclusion, passive smoking compromised bone neoformation in the defects and the LLLT protocol was not adequate to stimulate local osteogenesis.

Epithelial-stromal interactions in salivary glands of rats exposed to chronic passive smoking

OBJECTIVES Cigarette smoke leads to precancerous and cancerous lesions in the mouth even when the exposure is passive. The salivary glands are amongst the tissues exposed to the smoke but it is unclear whether or not passive cigarette exposure is related to detectable changes in these tissues. The objective of this study was to observe the tissue architecture of the parotid and submandibular glands in rats after passive cigarette exposure and to measure any changes that occurred. DESIGN Twenty Wistar rats were divided into 10 non-smoking animals and 10 animals exposed to cigarette smoke. After 6 months of smoke exposure samples were collected from both exposed and unexposed salivary glands for histological examination under both transmitted and polarized light microscopy. RESULTS Changes in the glands of exposed animals included involution of the cytoplasm and nucleus of the acinar cells and the presence of an inflammatory infiltrate. There was an abnormal accumulation of type I collagen in the stroma and an enlarged interacinar space filled with extracellular matrix. CONCLUSION Passive smoking led to substantial structural changes in the salivary glands which could significantly affect function.

Study of the osteoconductive capacity of hydroxyapatite implanted into the femur of ovariectomized rats.

Osteoporosis is a global public health that affects postmenopausal women due to the deficiency of estrogen, a hormone that plays an important role in the microarchitecture of bone tissue. Osteoporosis predisposes to pathological bone fracture that can be repaired by conventional methods. However, depending on the severity and quantity of bone loss, the use of autogenous grafts or biomaterials such as hydroxyapatite might be necessary. The latter has received increasing attention in the medical field because of its good biological properties such as osteoconductivity and biocompatibility with bone tissue. The objective of this study was to evaluate using histologic and radiographic analyses, the osteogenic capacity of hydroxyapatite implanted into the femur of rats with ovariectomy‐induced osteoporosis. Eighteen rats were divided into three groups with six animals in each: group nonovariectomized, bilaterally ovariectomized not receiving estrogen replacement therapy, and bilaterally ovariectomized submitted to estrogen replacement therapy. Defects were created experimentally in the distal epiphysis of the femur with a surgical drill and filled with porous hydroxyapatite granules. The animals were sacrificed 8 weeks after surgery. The volume of newly formed bone in the implant area was quantified by morphometrical methods. The results were analyzed by ANOVA followed by the Tukey test (P < 0.05). The hydroxyapatite granules showed good radiopacity. Histological analysis revealed less quantity of newly formed bone in the ovariectomized group not submitted to hormone replacement therapy. In conclusion, bone neoformation can be expected even in bones compromised by estrogen deficiency, but the quantity and velocity of bone formation are lower. Microsc. Res. Tech., 2011.

Hydroxyapatite and a New Fibrin Sealant Derived from Snake Venom as Scaffold to Treatment of Cranial Defects in Rats

Biomaterials are used as a promising alternative to bone grafts, including bioceramics whose composition resembles that of bone and fibrin sealants due to their hemostatic properties. The objective was to evaluate the repair of cranial defects in 40 rats, grafted with hydroxyapatite and a new fibrin sealant derived from snake venom. The animals were divided into four groups: C (control, no graft); Ha (hydroxyapatite); FS (fibrin sealant), and HaFS (hydroxyapatite and fibrin sealant). The animals were euthanized 2 and 6 weeks after surgery and wound area were submitted to analysis. After 2 weeks, immature bone was formed from the borders of the defect and in groups Ha and HaFS, few hydroxyapatite particles were surrounded by new bone. After 6 weeks, the new bone was mature and surrounded several hydroxyapatite particles, without connective tissue interposition and the volume of new bone was higher in HaFS group. The hydroxyapatite in combination with the new fibrin sealant accelerates bone repair.

Feasibility study of collagen membranes derived from bovine pericardium and intestinal serosa for the repair of cranial defects in ovariectomised rats.

The indication of biomaterials has increased substantially in the regenerative therapy of bone defects. However, in addition to evaluating the physicochemical properties of biomaterials, the quality of the recipient tissue is also essential for the osseointegration of implants, as abnormalities in bone metabolism, such as gonadal hormone deficiency, can influence bone healing. This study evaluated the osteoregenerative capacity of collagen membranes derived from bovine pericardium and intestinal serosa in the repair of cranial defects in ovariectomised rats. Thirty female Wistar rats were submitted to surgical creation of a 5-mm cranial bone defect. The rats were divided into a control group (not ovariectomised) and an ovariectomised group. The non-ovariectomised group was divided into three subgroups: control (G1) in which the defect was not filled with the biomaterial, and two subgroups (G2 and G3) that received the bovine pericardium- and serosa-derived collagen membranes, respectively. The ovariectomised group was divided into the same subgroups (G4, G5, and G6). The animals were sacrificed 8 weeks after surgery. The calvaria were removed for macroscopic and radiographic photodocumentation and processed for histomorphometric analysis of bone healing at the surgical site. Macroscopic, radiological, and microscopic analyses demonstrated the biocompatibility of the implanted collagen membranes, as indicated by the absence of infiltration and signs of inflammation at the surgical site. Histologically, discrete immature bone neoformation projecting from the margins of the defect was observed at the surgical site in ovariectomised groups when compared to the non-ovariectomised groups. The volume of newly formed bone was significantly higher in the non-ovariectomised groups (G1: 7.83%±1.32; G2: 21.33%±1.96; and G3: 22.83%±0.98) compared to the respective ovariectomised subgroups (G4: 3.16%±0.75; G5: 16.83%±0.98; and G6: 16.16%±0.75), thus demonstrating the deleterious effects of ovariectomy on bone homeostasis. Higher volumes of newly formed bone were observed in the groups receiving the membrane grafts (G2, G3, G5, and G6) compared to the control groups (G1 and G4). In conclusion, the bilateral ovariectomy compromises the ability to repair bone lesions grafted with osteoconductive biomaterials as in the case of collagen membranes derived from both bovine pericardium and intestinal serosa.

Use of a New Fibrin Sealant and Laser Irradiation in the Repair of Skull Defects in Rats

This study evaluated the osteogenic capacity of a new fibrin sealant (FS) combined with bone graft and laser irradiation in the bone repair. Defects were created in the skull of 30 rats and filled with autogenous graft and FS derived from snake venom. Immediately after implantation, low-power laser was applied on the surgical site. The animals were divided in: control group with autogenous graft (G1), autogenous graft and laser 5 J/cm2 (G2), autogenous graft and laser 7 J/cm2 (G3), autogenous graft and FS (G4), autogenous graft, FS and laser 5 J/cm2 (G5), autogenous graft, FS and laser 7 J/cm2 (G6). The animals were sacrificed 6 weeks after implant. Results showed absence of inflammatory infiltrate in the bone defect. New bone formation occurred in all groups, but it was most intense in G6. Thus, the FS and laser 7 J/cm2 showed osteoconductive capacity and can be an interesting resource to be applied in surgery of bone reconstruction.

Osteoconductive Capacity of Hydroxyapatite Implanted Into the Skull of Diabetics

AbstractDiabetes mellitus can cause various diseases, including loss of bone mineral density as a characteristic manifestation of osteoporosis. In this condition, bone is more vulnerable to pathologic fractures that can be treated by implantation of biomaterial grafts. The aim of this study was to evaluate the osteogenic capacity of hydroxyapatite implanted into bone defects in the skull of nonobese diabetic mice. Fifteen nonobese diabetic mice were divided into 3 groups: control (nondiabetic), spontaneously diabetic, and spontaneously diabetic receiving insulin replacement applied subcutaneously into the dorsum. Defects were created experimentally in the skull with a surgical bur and filled with hydroxyapatite granules. The animals were killed 4 weeks after surgery, and samples were obtained for analysis. Quantitative methods were used for measurement of the new bone formation. Data were analyzed by analysis of variance followed by the Tukey test (P < 0.05). Radiographic results showed good radiopacity of the hydroxyapatite; however, radiolucent spots were seen between the hydroxyapatite granules in the diabetic groups, indicating infiltration of connective tissue. Microscopic results showed projections of newly formed bone from the margin of bone defect toward the implant. The quantity of newly formed bone was significantly higher (P < 0.05) than that observed in the diabetic groups. The recipient area of diabetic groups contained a larger amount of connective tissue as demonstrated by radiographic analyses. In conclusion, the osteogenesis guided by the properties of hydroxyapatite may even occur in bone suffering from the effects of diabetes, but the quantity of newly formed bone is lower, and the process is slower.

Osseointegration of Hydroxyapatite Implants in Rat Tibial Defects with Sciatic Nerve Injury

El metabolismo oseo esta influenciado por diferentes factores y la actividad muscular como un estimulador de la plasticidad osea. Condiciones tales como lesiones nerviosas pueden comprometer la fisiologia osea debido a la inactividad muscular. Estudios previos han demostrado que el dano nervioso reduce la sustancia P y el peptido relacionado con el gen de la calcitonina, tambien conocidos como neuropeptidos que pueden tener un papel clave en la cicatrizacion osea. Este estudio evaluo la oseointegracion de los implantes de hidroxiapatita en defectos tibiales de ratas sometidas a la seccion del nervio ciatico unilateralmente. Doce ratas Wistar se dividieron en dos grupos (G1 y G2). En G1, el nervio ciatico se dejo intacto y en el G2 el nervio ciatico izquierdo fue completamente seccionado. Un defecto oseo tibial fue creado experimentalmente en ambos grupos y se relleno con granulos de hidroxiapatita. Los animales se sacrificaron 2 meses despues de la implantacion y las muestras fueron sometidas a inspeccion macroscopica y el analisis histologico. Se observo buena radiopacidad de los granulos de hidroxiapatita y definicion radiografica del defecto oseo. El analisis histologico revelo neoformacion osea adyacente a los granulos de hidroxiapatita en G1 y, en menor medida en G2, donde la proliferacion de tejido conectivo predomino en el sitio de implante. La neoformacion osea estimulada por hidroxiapatita en defectos oseos se puede esperar incluso en animales con paralisis de los miembros producto de una lesion nerviosa; sin embargo, la formacion de hueso se produce a menor velocidad en estos animales y su volumen es menor.

Evaluation of the bone repair in defects grafted with hydroxyapatite and collagen membrane combined with laser therapy in rats

Bone regeneration is the result of cellular events such as osteogenesis and neovascularization. However, implantation of autogenous grafts may be necessary in cases of bone mass loss due to high impact trauma. The disadvantages of the latter approach include morbidity of the donor area. Biomaterials represent an alternative for bone restoration. The most widely used compounds are collagen or hydroxyapatite membranes because of their biocompatibility and osteoconductivity. Laser therapy has been applied in combination with these implants to accelerate bone regeneration. The objective of this study was to evaluate the effects of low-level laser therapy (LLLT) on the healing of rat left tibial bone defects filled with hydroxyapatite or collagen membrane. Twenty rats were used. Surgical bone defects were created in the proximal third of the left tibia, and the animals were divided into four groups according to treatment: animals receiving hydroxyapatite implants (group H), animals receiving collagen implants ...