Ricardo Junqueira Del Carlo

Células-tronco mesenquimais

Of all the stem cells studied so far, the mesenchymal stem cells (MSC) stand out for their high plasticity and capacity of generating mesodermal and non-mesodermal tissues. In addition, immunomodulatory and immunosuppressive features that expand possibilities for therapeutic use are present in these cells. A variety of pro and anti-inflammatory cytokines and growth factors are secrete for MSC and provide a modulation of inflammatory response, re-establishment of vascular supply and adequate repair of the tissue, contributing to tissue homeostasis under physiologic conditions. Therefore, they can induce secretion of soluble factors that stimulate their differentiation by other cells present at the niches tissue, promoting the repair process. Cell therapy using MSC is a promises therapeutic alternative, but understanding the biology of these cells is still under construction. The aim of the article is to conduct a short review of these undifferentiated mesenchymal cells.

Differentiation of adipose tissue-derived mesenchymal stem cells into cardiomyocytes

BACKGROUND Cardiomyocytes have small potential for renovation and proliferation in vivo. Consequently, the heart muscle has limited capacity of self-renewal. Mesenchymal stem cells (MSC) therapy, as well as MSC differentiated into cardiomyocytes, has been used in the attempt to minimize the effects of ischemic-hypoxic lesions and those affecting the electrical conduction system of the heart. OBJECTIVE The present study compared three distinct protocols for induced differentiation of MSC into cardiomyocytes aimed at finding a viable method for producing a large number of functional cells expressing cardiomyogenic phenotype. METHODS Mesenchymal stem cells were obtained from the adipose tissue of young transgenic Lewis rats expressing green fluorescent protein (GFP), and submitted to three distinct differentiation-inducing media: 1) Planat-Bérnard, 2) 5-azacytidine, and 3) Planat-Bérnard + 5-azacytidine; further, these cells were identified based on the expression of cardiac cell markers. RESULTS All three protocols detected the expression of sarcomeric-alpha-actinin protein in the exoskeleton of cells, expression of connexin-43 in the nuclear and cytoplasmic membrane, and formation of gap junctions, which are necessary for electrical impulse propagation in the myocardium. However, no spontaneous cell contraction was observed with any of the tested protocols. CONCLUSION Induction with 5-azacytidine provided an effective cadiomyogenic cellular differentiation similar to that obtained with Planat-Bénard media. Therefore, 5-azacytidine was the method of choice for being the simplest, fastest and lowest-cost protocol for cell differentiation.

Association of mesenchymal stem cells with platelet rich plasma on the repair of critical calvarial defects in mice

PURPOSE To evaluate the effects of mesenchymal stem cells (MSC) from eight mice C57BL/6 gfp(+) bone marrows expanded in cultures associated with platelets rich plasma (PRP) deriving from another eight mice, in the repair of critical defects in calvarial bone produced in twenty-four adult isogenic mice C57BL/6. METHODS The animals were submitted to a cranial defect of 6.0mm in diameter and divided into two equal experimental groups. Control group did not receive treatment and the treated group received a MSC pellet containing 1.0 x 10(7) cells/mL associated with 50.0 µL of plasma gel containing 1.0 x 10(9) autologous platelets within the defect. RESULTS In the treated group was observed process of angiogenesis and bone repair better than control group. CONCLUSION Mesenchymal stem cells derived from bone marrow of C57BL/6 gfp(+) mice associated with PRP gel applied in bone critical defects produced in calvarial contributes positively to the process of bone repair.

Comparison of in vivo properties of hydroxyapatite-polyhydroxybutyrate composites assessed for bone substitution.

This study compared 3 composites made of hydroxyapatite and polyhydroxybutyrate to determine their biocompatibility, osteointegration, and osteoconduction in rabbits bone defects. Hydroxyapatite-polyhydroxybutyrate was tested in a 10:90 vol/vol ratio for composite 1, 25:75 for composite 2, and 50:50 for composite 3, whereas defects in the control group remained unfilled. Limbs were clinically and radiographically evaluated, and samples were taken 8, 45, and 90 days after surgery for histologic and histomorphometrical analysis. No significant difference was found in the clinical parameters: pain, lameness, dehiscence, infection, and edema among the 4 groups. No signs of osteolysis or periosteal reaction were observed. Bone regeneration in groups 1, 2, and 3 occurred histomorphologically similar to the control group. No inflammatory infiltrate was present on any date of observation. More bone than connective tissue was significantly found in the defects of the 3 groups on all observation dates, and the percentage of bone tissue in the interface was similar for composites 2 and 3. Bone and connective tissue were observed in pores of composites 2 and 3. Osteoclasts were found in the bone-composite interface, and multinucleated giant cells were seen on the surface of composite fragments, found among mesenchymal tissue on the 45th and 90th days. No significant difference was observed in osteoclast number among the 4 groups. In summary, composites were considered biocompatible and able to integrate to bone. They also showed signs of biodegradability, and composites 2 and 3 were osteoconductive, the first displaying best characteristics for bone substitution.

Plasma rico em plaquetas associado ou não ao osso esponjoso autógeno no reparo de falhas ósseas experimentais

The present study evaluated autogenous platelet rich plasmas (PRP) influence on the reparation process of four bone defects made on rabbits skull, associated or not to autogenous bone graft (EOE). Defect I received PRP only; defect II received 3mg of EOE only; defect III received EOE associated to PRP; defect IV was left to heal naturally, serving as control. After each surgery the animals were randomly divided into three groups that were euthanized at 30, 60 and 90 days. In the mesoscopic evaluation bone ingrowth started from the defects borders to the center and from the bottom to the surface for all observation times on the control (VI) and PRP only (I) groups. In the groups treated with EOE only (II) and EOE associated to PRP (III) new bone was observed in the center of the defects. Radiographic analysis showed greater central radiopacity for groups treated with EOE only (II) and EOE associated to PRP (III) at all observation times. Microscopically in the group treated with EOE associated to PRP (III) at 30 days the graft was indistinguishable from new bone present on the border of the defect, associated to a moderate quantity of a very vascularized and cellular fibrous connective tissue. This tissue showed an extracelular eosinophilic amorphous foamy material, associated to an inflammatory process constituted by lymphocytes and in less number by macrophages and multinucleated giant cells that may have negatively influenced early bone formation. At 60 and 90 days occasional spots of lymphocytic inflammation were observed. Both treatments, PRP associated or not to EOE, were similar for the bone ingrowth at the final time of observation; the graft used alone determined early bone reparation and thromboplastine used for the platelet gel formation incited a foreign body-like reaction that acted negatively on the initial reparation.

Eletroterapia no processo de reparação da superfície articular de coelhos

The effects of postooperative electrotherapy in the repair of full-thickness osteochondral defects in the troclear groove of 36 adult rabbits were evaluated histologically. In the control group, granulation tissue was present two weeks post-operatively (PO); hyaline cartilage was observed at four and at seven weeks PO; subchondral bone regeneration was limited to the deeper portions of the defects at seven weeks PO. In group I (stimulated daily), granulation tissue presented abundant vascularization at two weeks PO and the repair tissue remained undifferentiated superficially, with formation of new subchondral bone up to the level of the original osteochondral junction, at seven weeks PO. The animals in group II (stimulated every other day), responded in two ways: through repair with hyaline cartilage, with reduced osteogenesis, similar to control group; or undifferentiated tissue, with an intense osteogenesis, as in group I. The postoperative electrical stimulation inhibited the tissue differentiation into hialine cartilage. On the other hand, endochondral ossification was accelerated, with complete regeneration of subchondral bone, in treated animals. In view of the importance of the subchondral bone for the integrity of the repair tissue, electrical stimulation is benefic in the initial fase (four weeks) of osteochondral defects repair.

Treatment of critical defects produced in calvaria of mice with mesenchymal stem cells

Mesenchymal stem cells (MSC) are present in specialized niches in perivascular regions of adult tissues and are able to differentiate into various cell types, such as those committed to repairing. Bone marrow derived MSC from eight young mice C57BL/ 6 gfp(+) were expanded in culture for repairing critical defects in calvarial bone produced in twenty-four young isogenic adult C57BL/6 mice. The animals were subjected to a cranial defect of 6.0mm diameter and divided into two equal experimental groups. Control group did not receive any treatment and the treated group received a MSC pellet containing 1.0 x 10(7) cells/mL into the defects. The group treated with MSC showed increased angiogenesis and amount of new bone deposited on the defect limits than that observed in the control group. The results demonstrated that transplantation of bone marrow-derived MSC of C57BL/6 gfp(+) mice to bone critical defects produced in mice calvarial contributes positively to the bone repair process. MSC presets ability to influence the correct functioning of osteoblasts, increases the amount of mobilized cells for the repairing process, speeds up growth, and increases deposition of bone matrix.

Polímero derivado de mamona acrescido de cálcio, associado ou não à medula óssea autógena na reparação de falhas ósseas

In order to evaluate tissue repair after the use of castor oil polymer implant additioned with 40% sodium carbonate, isolated or associated to autogenous bone marrow in heterotopic site and in experimental bone gaps in radii of rabbits, 30 White New Zealand rabbits were submitted to bilateral radial ostectomy. In 15 rabbits the bone gap of the right side was filled with polymer cylinders (group P) of similar size of the gaps; the remaining rabbits received autogenous bone marrow with the implant (group M). The bone defects of the left limb did not receive any treatment and served as control. Six rabbits received 6 implants in the Rectus abdominus muscle (heterotopic site) and in three of these rabbits the implants was associated with bone marrow. In the radiographic study both groups presented increased radiopacity at the implant site without bone axis deviation or resorption of the receptor bone ends. Group P presented irregular calcification areas at the peripheral region and over the polymer. Group M presented a more intense, regular and precocious radiopacity pattern in relation to group P. In microscopical evaluation there was evidence of immature bone tissue formation tending to organize itself, isolated sprouts of neoformed bone over the polymer and its superficial pores. It was concluded that implant allows osteogenenesis and osteoconduction in bone gaps, and bone formation was progressive, especially when additioned bone marrow aspirate; there was capillary, perivascular tissue and osteoprogenitor cells migrating into the pores; with fibrovascular tissue permeating implant surface; implants incorporation was slowly and was found incomplete until 9 weeks; the implant was able to induce foreign body reaction without toxic or secondary reactions to its presence. In heterotopic and orthotopic sites, the implant was not able to induce bone formation, inciting mild foreign-body reaction with giant cells and surrounding fibrous layer.

Efeitos de tratamento combinado de alendronato de sódio, atorvastatina cálcica e ipriflavona na osteoporose induzida com dexametasona em ratas

The objective of this work was to study the influence of the combinations: biphosphonate (alendronate of sodium) + statin (calcic atorvastatin), biphosphonate (sodium alendronate) + flavonoid (ipriflavone) and statin (calcic atorvastatin) + flavonoid (ipriflavone) in female rats with osteoporosis induced by the glucocorticoid dexamethasone. The influence of the combinations of those drugs was observed through tests of the biochemical markers of bone remodeling, such as, levels of serum calcium and phosphorus and bone alkaline phosphatase and through histomorphometric exam, visualizing the bone trabecular density. Through the evaluation of the bone trabecular density it was possible to verify the clear increase in any group of treated animals. The combinations with biphosphonate presented high rates of bone tissue restoration, reaching higher values than those of normal animals. The results obtained with biochemical markers were not statistically significant and did not subsidies the diagnosis and the follow of the osteoporosis. However, the histomorphometric evaluation allowed us the static and dynamic analyses as well as detection of tissue alterations in the bone metabolic unit, particularly in the trabecular bone.

Avaliação histológica do processo de reparação da superfície articular de coelhos

The repairing process in the articular surface was evaluated microscopically in an osteochondral gap produced in the left troclear groove of 12 adult rabbits. It was observed that the granulation tissue wich initially filled this gap had diferentiated into hyaline cartilage by 4 weeks after surgery, presenting parcial reconstitution of subchondral bone excised at seven weeks following surgery. In some animals, cleft formation was observed between the repair tissue and adjacent bone, always where bone remodeling did not occured. In animals wich the bottom and lateral edges of the defect were formed by lamelar subchondral bone with few blood vessels, bone remodeling was not observed, having no attachment of the repair tissue to the adjacent bone. The results show the importance of complete removal of the lamelar subcondral bone during the surgical procedures in the articular surface, in order to allow better blood supply from the subjacent cancellous bone, better bone remodeling, and attachment of the repair tissue to the bottom of the defect.