Culture of rat mesenchymal stem cells on PHBV-PCL scaffolds: analysis of conditioned culture medium by FT-Raman spectroscopy / Cultivo de células-tronco mesenquimais de rato sobre arcabouços de PHBV-PCL: análise do meio de cultura condicionado por espectroscopia de FT-Raman

Abstract Mesenchymal stem cells (MSCs) have great potential for application in cell therapy and tissue engineering procedures because of their plasticity and capacity to differentiate into different cell types. Given the widespread use of MSCs, it is necessary to better understand some properties related to osteogenic differentiation, particularly those linked to biomaterials used in tissue engineering. The aim of this study was to develop an analysis method using FT-Raman spectroscopy for the identification and quantification of biochemical components present in conditioned culture media derived from MSCs with or without induction of osteogenic differentiation. All experiments were performed between passages 3 and 5. For this analysis, MSCs were cultured on scaffolds composed of bioresorbable poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and poly(ε-caprolactone) (PCL) polymers. MSCs (GIBCO®) were inoculated onto the pure polymers and 75:25 PHBV/PCL blend (dense and porous samples). The plate itself was used as control. The cells were maintained in DMEM (with low glucose) containing GlutaMAX® and 10% FBS at 37oC with 5% CO2 for 21 days. The conditioned culture media were collected and analyzed to probe for functional groups, as well as possible molecular variations associated with cell differentiation and metabolism. The method permitted to identify functional groups of specific molecules in the conditioned medium such as cholesterol, phosphatidylinositol, triglycerides, beta-subunit polypeptides, amide regions and hydrogen bonds of proteins, in addition to DNA expression. In the present study, FT-Raman spectroscopy exhibited limited resolution since different molecules can express similar or even the same stretching vibrations, a fact that makes analysis difficult. There were no variations in the readings between the samples studied. In conclusion, FT-Raman spectroscopy did not meet expectations under the conditions studied.

Resumo As células-tronco mesenquimais (MSCs) possuem grande potencial para aplicação em procedimentos terapêuticos ligados a terapia celular e engenharia de tecidos, considerando-se a plasticidade e capacidade de formação em diferentes tipos celulares por elas. Dada a abrangência no emprego das MSCs, há necessidade de se compreender melhor algumas propriedades relacionadas à diferenciação osteogênica, particularmente liga à biomateriais usados em engenharia de tecidos. Este projeto objetiva o desenvolvimento de uma metodologia de análise empregando-se a FT-Raman para identificação e quantificação de componentes bioquímicos presentes em meios de cultura condicionados por MSCs, com ou sem indução à diferenciação osteogênica. Todos os experimentos foram realizados entre as passagens 3 e 5. Para essas análises, as MSCs foram cultivadas sobre arcabouços de polímeros biorreabsorvíveis de poli (hidroxibutirato-co-hidroxivalerato) (PHBV) e o poli (ε-caprolactona) (PCL). As MSCs (GIBCO®) foram inoculadas nos polímeros puros e na mistura 75:25 de PHBV / PCL (amostras densas e porosas). As células foram mantidas em DMEM (com baixa glicose) contendo GlutaMAX® e 10% de SFB a 37oC com 5% de CO2 por 21 dias. A própria placa foi usada como controle. Os meios de cultura condicionados foram coletados e analisadas em FT-Raman para sondagem de grupos funcionais, bem como possíveis variações moleculares associadas com a diferenciação e metabolismo celular. Foi possível discernir grupos funcionais de moléculas específicas no meio condicionado, como colesterol, fosfatidilinositol, triglicerídeos, forma Beta de polipeptídeos, regiões de amida e ligações de hidrogênio de proteínas, além da expressão de DNA. Na presente avaliação, a FT-Raman apresentou como uma técnica de resolução limitada, uma vez que modos vibracionais de estiramento próximos ou mesmo iguais podem ser expressos por moléculas diferente, dificultando a análise. Não houve variações nas leituras entre as amostras estudadas, concluindo-se que a FT-Raman não atendeu às expectativas nas condições estudadas.

Automated Assessment of Cell Infiltration and Removal in Decellularized Scaffolds - Experimental Study in Rabbits / Avaliação automatizada da infiltração e remoção celular em scaffolds descelularizados - Estudo experimental em coelhos

Abstract Objective Semiquantitative and automated measurement of nuclear material removal and cell infiltration in decellularized tendon scaffolds (DTSs). Method 16 pure New Zealand rabbits were used, and the gastrocnemius muscle tendon was collected bilaterally from half of these animals (16 tendons collected); 4 were kept as control and 12 were submitted to the decellularization protocol (DTS). Eight of the DTSs were used as an in vivo implant in the experimental rotator cuff tear (RCT) model, and the rest, as well as the controls, were used in the semiquantitative and automated evaluation of nuclear material removal. The eight additional rabbits were used to make the experimental model of RCT and subsequent evaluation of cellular infiltration after 2 or 8 weeks, within the DTS. Results The semiquantitative and automated analysis used demonstrated a removal of 79% of nuclear material (p< 0.001 and power > 99%) and a decrease of 88% (p < 0.001 and power >99%) in the area occupied by nuclear material after the decellularization protocol. On cell infiltration in DTS, an increase of 256% (p < 0.001 and power >99%) in the number of cells within the DTS was observed in the comparison between 2 and 8 weeks postoperatively. Conclusion The proposed semiquantitative and automated measurement method was able to objectively measure the removal of nuclear material and cell infiltration in DTS.

Resumo Objetivo Mensuração semiquantitativa e automatizada da remoção de material nuclear e da infiltração celular em scaffolds tendinosos descelularizados (STDs). Método Foram utilizados 16 coelhos Nova Zelândia puros, sendo o tendão do músculo gastrocnêmio coletado bilateralmente de metade destes animais (16 tendões coletados); 4 foram mantidos como controle e 12 foram submetidos ao protocolo de descelularização (STD). Dos STDs, 8 foram utilizados como implante in vivo no modelo experimental de lesão do manguito rotador (LMR) e os restantes, assim como os controles, foram utilizados na avaliação semiquantitativa e automatizada da remoção de material nuclear. Os oito coelhos adicionais foram utilizados na confecção do modelo experimental de LMR e posterior avaliação da infiltração celular após 2 ou 8 semanas, dentro do STD. Resultados A análise semiquantitativa e automatizada utilizada demonstrou uma remoção de 79% do material nuclear (p< 0,001 e poder > 99%) e uma diminuição de 88% (p< 0,001 e poder > 99%) na área ocupada por material nuclear após o protocolo de descelularização. Sobre a infiltração celular no STD, foi observado um aumento de 256% (p< 0,001 e poder > 99%) no número de células dentro do STD na comparação entre 2 e 8 semanas de pós-operatório. Conclusão O método de mensuração semiquantitativo e automatizado proposto foi capaz de mensurar objetivamente a remoção de material nuclear e a infiltração celular no STD.

The effect of scaffold hydroxyapatite derived from portunus pelagicus shell on the expression of fibroblast growth factor-2 (fgf-2) and bone morphogenetic proteins-2 (bmp -2) in the extraction sockets of cavia cobaya / El efecto del scaffold de hidroxiapatita derivada de portunus pelagicus sobre la expresión del factor de crecimiento de fibroblastos-2 (FGF-2) y las proteínas morfogenéticas óseas-2 (BMP-2) en los alvéolos dentarios de cavia cobaya

Objetive: To determine the expression of Fibroblast Growth Factor (FGF)-2 and Bone Morphogenetic Protein (BMP)-2 after application of scaffold hydroxyapatite from Rajungan crab shell (Portunus pelagicus) in the tooth extraction socket of Cavia cobaya. Material and Methods: This study used a post-test only control group design with 28 Cavia cobaya separated into two groups, control and treatment group. The left mandibular incisor was extracted, and socket preservation was conducted. A hydroxyapatite graft derived from crab shells was mixed with gelatin and eventually turned into a scaffold, which was afterward put into the extraction socket. After 7 days and 14 days, each group was terminated and examined using immunohistochemical staining to observe the expression of FGF-2 and BMP-2. One-Way Anova and Tukey HSD were used to examine the research data. Results: FGF-2 and BMP-2 expressions were observed higher in the group that received hydroxyapatite scaffold at the post-extraction socket than those in the group that did not receive hydroxyapatite scaffold. Conclusion: The application of a hydroxyapatite scaffold from Rajungan crab shell (Portunus pelagicus) to the tooth extraction socket can increase FGF-2 and BMP-2 expression.

Objetivo: Determinar la expresión del factor de crecimiento de fibroblastos (FGF)-2 y la proteína morfogenética ósea (BMP)-2 después de la aplicación de hidroxiapatita de andamio de caparazón de cangrejo Rajungan (Portunus pelagicus) en el alvéolo de extracción dental de Cavia cobaya. Material y Métodos: Este estudio utilizó un diseño de grupo de control solo posterior a la prueba con 28 Cavia cobaya separados en dos grupos, grupo de control y grupo de tratamiento. Se extrajo el incisivo mandibular izquierdo y se realizó la preservación del alvéolo. Un injerto de hidroxiapatita derivado de caparazones de cangrejo se mezcló con gelatina y se convirtió en un andamio, que luego se colocó en el alvéolo de extracción. Después de 7 días y 14 días, se terminó cada grupo y se examinó mediante tinción inmunohistoquímica para observar la expresión de FGF-2 y BMP-2. Se utilizaron One-Way Anova y Tukey HSD para examinar los datos de la investigación. Resultados: Las expresiones de FGF-2 y BMP-2 se observaron más altas en el grupo que recibió la estructura de hidroxiapatita en el alvéolo posterior a la extracción que en el grupo que no recibió la estructura de hidroxiapatita. Conclusión: La aplicación de un andamio de hidroxiapatita de caparazón de cangrejo Rajungan (Portunus pelagicus) al alvéolo de extracción dental puede aumentar la expresión de FGF-2 y BMP-2.

Células madre en odontología: nuevas perspectivas / Stem cells in dentistry: new perspectives

En el campo de la odontología, prevalecen actualmente alternativas terapéuticas con una filosofía conservadora. Sin embargo, con el advenimiento de los tratamientos con células madre (CM), se amplían las posibilidades terapéuticas, que buscan la combinación y el equilibrio entre la intervención tradicional y las posibilidades de reposición de estructuras anatómicas dañadas, a través de la regeneración de tejidos utilizando células madre o sus derivados (AU)

In the dentistry field, therapeutic alternatives with a conservative philosophy currently prevail. However, with the advent of stem cell (SC) treatments, therapeutic possibilities are expanding, seeking a combination and balance between traditional intervention and the pos- sibility of replacing damaged anatomical structures through tissue regeneration, using stem cells or their derivatives (AU)

Decoloración coronaria inducida por la utilización de Biodentine y MTA blanco en procedimientos de regeneración pulpar: estudio ex vivo / Coronal discoloration induced by the use of Biodentine and white MTA in pulp regeneration procedures: an ex-vivo study

Se comparó el grado de decoloración de la corona clínica en piezas dentarias anteriores humanas ex-traídas, sometidas a procedimientos de regeneración endodóntica, utilizando MTA blanco o sustituto bioac-tivo de la dentina (Biodentine) como barrera cervical, en presencia de coágulo sanguíneo como andamio. En total se prepararon 24 piezas dentarias anterio-res superiores humanas que fueron divididas en dos grupos control (GC) y dos grupos experimentales (GE). Cada uno incluía 6 piezas dentarias. En los GE se colo-có sangre humana fresca en el interior del conducto, y se confeccionó una barrera de Biodentine (GE3) o MTA (GE4). En los GC se colocó una torunda de algo-dón estéril saturada con solución fisiológica estéril, y se confeccionó una barrera de Biodentine (GC1) o MTA (GC2). El color se evaluó de acuerdo con el espa-cio de color CIE L* a* b* utilizando imágenes fotográfi-cas digitales estandarizadas en dos puntos de tiempo: día 0 (T0) y día 35 (T35). La descripción de los datos in-cluyó mediana (Md), primer cuartil (Q1), tercer cuartil (Q3), media y desviación estándar (DE). Se emplea-ron las pruebas de los rangos con signo de Wilcoxon (RSW) y ANOVA de una vía; p < 0,05 fue considerado significativo. Cuando se comparó ∆E se observaron diferencias significativas entre GC1 y el resto de los materiales (p < 0,05). ∆E fue menor en GC1 (media ± DE; 2,1 ± 1,6) que en los grupos restantes que no pre-sentaron diferencias significativas entre sí. En base a estos resultados, la estabilización del coágulo san-guíneo, como así la limpieza de la cavidad previa a la colocación del Biodentine y el uso de barreras cervi-cales, es imprescindible para evitar la coloración de la corona clínica (AU)

Discoloration remains an unfavorable complication of otherwise successful regenerative endodontic procedure of immature teeth with necrotic pulp. Objective: Compare the degree of discoloration of extracted human teeth after regenerative endodontic procedures, using MTA or Biodentine as a cervical barrier with a blood clot as a scaffold. In total 24 human upper anterior teeth were prepared and divided into two control groups (CG) and two experimental groups (EG). In the EG, fresh human blood was placed inside the root and a Biodentine (GE3) or MTA (GE4) barrier was made in. A sterile cotton swab saturated with sterile physiological solution was placed in the GCs and a Biodentine (GC1) or MTA (GC2) barrier was made in. The color was evaluated according to the CIE L* a* b* color space using standardized digital photographic images at two time points: day 0 (T0) and day 35 (T35). The description of the data included median (Md), first quartile (Q1), third quartile (Q3), means, and standard deviation (SD). Wilcoxon signed rank tests (RSW) and one-way ANOVA were used. p < 0.05 was considered significant. When ∆E was compared, significant differences were observed between GC1 and the rest of the materials (p < 0.05). ∆E was lower in CG1 (mean ± SD; 2.1 ± 1.6) than in the remaining groups, which did not present significant differences between them. There was no significant difference between tooth discolorations with materials in the presence of blood. However, in the absence of blood, Biodentine exhibited less tooth discoloration than MTA (AU)

Recovery of motor function in rats with complete spinal cord injury following implantation of collagen/silk fibroin scaffold combined with human umbilical cord-mesenchymal stem cells

SUMMARY OBJECTIVE: This study aimed to assess the effect of the collagen/silk fibroin scaffolds seeded with human umbilical cord-mesenchymal stem cells on functional recovery after acute complete spinal cord injury. METHODS: The fibroin and collagen were mixed (mass ratio, 3:7), and the composite scaffolds were produced. Forty rats were randomly divided into the Sham group (without spinal cord injury), spinal cord injury group (spinal cord transection without any implantation), collagen/silk fibroin scaffolds group (spinal cord transection with implantation of the collagen/silk fibroin scaffolds), and collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group (spinal cord transection with the implantation of the collagen/silk fibroin scaffolds co-cultured with human umbilical cord-mesenchymal stem cells). Motor evoked potential, Basso-Beattie-Bresnahan scale, modified Bielschowsky's silver staining, and immunofluorescence staining were performed. RESULTS: The BBB scores in the collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group were significantly higher than those in the spinal cord injury and collagen/silk fibroin scaffolds groups (p<0.05 or p<0.01). The amplitude and latency were markedly improved in the collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group compared with the spinal cord injury and collagen/silk fibroin scaffolds groups (p<0.05 or p<0.01). Meanwhile, compared to the spinal cord injury and collagen/silk fibroin scaffolds groups, more neurofilament positive nerve fiber ensheathed by myelin basic protein positive structure at the injury site were observed in the collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group (p<0.01, p<0.05). The results of Bielschowsky's silver staining indicated more nerve fibers was observed at the lesion site in the collagen/silk fibroin scaffolds + human umbilical cord-mesenchymal stem cells group compared with the spinal cord injury and collagen/silk fibroin scaffolds groups (p<0.01, p< 0.05). CONCLUSION: The results demonstrated that the transplantation of human umbilical cord-mesenchymal stem cells on a collagen/silk fibroin scaffolds could promote nerve regeneration, and recovery of neurological function after acute spinal cord injury.

Three-dimensional printed poly (L-lactide) and hydroxyapatite composite for reconstruction of critical bone defect in rabbits

ABSTRACT Purpose To use a 3D printed poly (L-lactide) acid (PLLA) and hydroxyapatite (HA) composite as a bone substitute for reconstruction of a critical bone defect in the radius of rabbits. Methods A 1.5 cm ostectomy was performed in the radial diaphysis of 60 New Zealand white rabbits. The rabbits were divided into three groups according to surgical treatment of the bone defect (group I - control, group II - bone graft, group III - 3D PLLA). Each group was divided into four subgroups with different radiographic and histopathologic evaluation times (T1 - 15 days, T2 - 30 days, T3 - 60 days, T4 - 90 days). Results The implant group had greater clinically lameness (p = 0.02), edema (p = 0.007), pain (p = 0.04) and more complications at the surgical site (p = 0.03). Histologically, this group showed greater congestion (p = 0.04), hemorrhage (p = 0.04) and inflammation. Osteogenesis was microscopically similar between days (p = 0.54) and treatments (p = 0.17), even though radiographically, more effective bone healing occurred in the graft group (II), with more callus and bone bridge formation. Conclusions The customization of a 3D PLLA/HA scaffold was successful. However, in animals receiving the polymer-ceramic composite less bone callus and bone bridge was formed compared to the graft group.

Quantification of adhesion of mesenchymal stem cells spread on decellularized vein scaffold

ABSTRACT Purpose: To evaluate methods that improve adipose-derived stem cells (ASCs) population in decellularized biological venous scaffold for tissue engineering in blood vessels, a model in rabbits. Methods: The ASC was expanded until the third passage. Inferior vena cava (IVC) was submitted to the decellularization process using 1% sodium dodecyl sulfate (SDS) or 2% sodium deoxycholate (SD) to compose 12 study groups (G): pure SD or SDS, exposed or not to 1% TritonX-100 (TX-100) and exposed or not to poly-l'lysine and laminin (PL). Scaffolds were covered with 1 × 105 or 1 × 106 ASCs diluted in 10 μL Puramatrix™. The histological analysis was done by cell counting in hematoxylin and eosin (HE) and nuclei count in immunofluorescence (IF) with 4',6-Diamidine-2'-phenylindole dihydrochloride (DAPI). Results: The study of groups in HE and IF showed similar results. For both analyses,IVC-SD-1 × 106 ASC and IVC-SD-PL-1 × 106 ASC provided the best results. The IF technique showed better sensitivity than HE, with a weak agreement between them. Conclusions: Decellularizing agent and the number of ASC influence scaffolds cellularization response and the best protocols as those ones using SD with or without the addition of PL.

Comparison of two different biomaterials in the bone regeneration (15, 30 and 60 days) of critical defects in rats

ABSTRACT Purpose To evaluate and compare two types of different scaffolds in critical bone defects in rats. Methods Seventy male Wistar rats (280 ± 20 grams) divided into three groups: control group (CG), untreated animals; biomaterial group 1 (BG1), animals that received the scaffold implanted hydroxyapatite (HA)/poly(lactic-co-glycolic) acid (PLGA); and biomaterial group 2 (BG2), animals that received the scaffolds HA/PLGA/Bleed. The critical bone defect was induced in the medial region of the skull calotte with the aid of an 8-mm-diameter trephine drill. The biomaterial was implanted in the form of 1.5 mm thick scaffolds, and samples were collected after 15, 30 and 60 days. Non-parametric Mann-Whitney test was used, with the significance level of 5% (p ≤ 0.05). Results Histology revealed morphological and structural differences of the neoformed tissue between the experimental groups. Collagen-1 (Col-1) findings are consistent with the histological ones, in which BG2 presented the highest amount of fibers in its tissue matrix in all evaluated periods. In contrast, the results of receptor activator of nuclear factor kappa-Β ligand (Rank-L) immunoexpression were higher in BG2 in the periods of 30 and 60 days, indicating an increase of the degradation of the biomaterial and the remodeling activity of the bone. Conclusions The properties of the HA/PLGA/Bleed scaffold were superior when compared to the scaffold composed only by HA/PLGA.

Biomaterials for bone regeneration: an orthopedic and dentistry overview

Because bone-associated diseases are increasing, a variety of tissue engineering approaches with bone regeneration purposes have been proposed over the last years. Bone tissue provides a number of important physiological and structural functions in the human body, being essential for hematopoietic maintenance and for providing support and protection of vital organs. Therefore, efforts to develop the ideal scaffold which is able to guide the bone regeneration processes is a relevant target for tissue engineering researchers. Several techniques have been used for scaffolding approaches, such as diverse types of biomaterials. On the other hand, metallic biomaterials are widely used as support devices in dentistry and orthopedics, constituting an important complement for the scaffolds. Hence, the aim of this review is to provide an overview of the degradable biomaterials and metal biomaterials proposed for bone regeneration in the orthopedic and dentistry fields in the last years.

Aplicaciones de la impresión 3D en cirugía plástica reconstructiva / Use of 3D printing in reconstructive plastic surgery

Resumen La impresión 3D es una tecnología interesante en constante evolución. También conocida como manufactura aditiva, consiste en la conversión de diseños digitales a modelos físicos mediante la adición de capas sucesivas de material. En años recientes, y tras el vencimiento de múltiples patentes, diversos campos de las ciencias de la salud se han interesado en sus posibles usos, siendo la cirugía plástica una de las especialidades médicas que más ha aprovechado sus ventajas y aplicaciones, en especial la capacidad de crear dispositivos altamente personalizados a costos accesibles. Teniendo en cuenta lo anterior, el objetivo del presente artículo es describir los usos de la impresión 3D en cirugía plástica reconstructiva a partir de una revisión de la literatura. Las principales aplicaciones de la impresión 3D descritas en la literatura incluyen su capacidad para crear modelos anatómicos basados en estudios de imagen de pacientes, que a su vez permiten planificar procedimientos quirúrgicos, fabricar implantes y prótesis personalizadas, crear instrumental quirúrgico para usos específicos y usar biotintas en ingeniería tisular. La impresión 3D es una tecnología prometedora con el potencial de implementar cambios positivos en la práctica de la cirugía plástica reconstructiva en el corto y mediano plazo.

Abstract 3D printing is an interesting technology in constant evolution. Also known as additive manufacturing, it consists of the conversion of digital designs into physical models by successively adding material layer by layer. In recent years, and after the expiration of multiple patents, several fields of health sciences have approached this type of technology, plastic surgery being one of the medical specialties that has taken advantage of its benefits and applications, especially the ability to create highly customized devices at low costs. With this in mind, the objective of this work is to describe the uses of 3D printing in reconstructive plastic surgery based on a literature review. The main applications of 3D printing described in the literature include its ability to create anatomical models based on patient imaging studies, which in turn allow planning surgical procedures, manufacturing custom implants and prostheses, creating surgical or instrumental simulators, and using bioinks in tissue engineering. 3D printing is a promising technology with the potential to cause positive changes in the field of reconstructive plastic surgery in the short and medium term.

Tratamiento del Diente Permanente Necrótico. Un Cambio de Paradigma en el Campo de la Endodoncia / Treatment of the Necrotic Permanent Tooth. A Paradigm Shift in Endodontics

RESUMEN: El tratamiento de dientes inmaduros necróticos es hoy un gran desafío clínico. La ausencia de cierre del ápice y el reducido grosor de las paredes de la dentina hacen que el tratamiento endodóntico del diente sea difícil e impredecible. Tradicionalmente, estos dientes han sido tratados con apexificación y obturación del canal radicular, sin embargo, con este tratamiento el diente permanece desvitalizado y con paredes de dentina frágiles y cortas, lo que compromete su pronóstico. La endodoncia regenerativa, por el contrario, busca revitalizar el diente e inducir una maduración de la raíz, y se basa en la utilización de las células madre mesenquimales presentes en la región periapical, los factores de crecimiento presentes en la dentina y un andamio que permite el crecimiento de tejido nuevo al interior del canal. Los resultados clínicos son alentadores, ya que en general existe maduración de la raíz y revascularización del diente, sin embargo, el tejido neoformado es tejido de tipo reparativo y, a excepción de estudios ocasionales, no se ha observado regeneración de dentina y pulpa. La endodoncia regenerativa se originó para tratar dientes inmaduros necróticos. Sin embargo, recientemente, estudios preliminares han expandido la aplicación de la endodoncia regenerativa a dientes maduros necróticos, es decir, en pacientes adultos. Los resultados clínicos son positivos y similares a los del diente inmaduro, si n embargo, la investigación referente a la revitalización de dientes maduros se encuentra en etapas tempranas y requiere de un mayor nivel de evidencia antes de ser ofrecida sistemáticamente como terapia a pacientes adultos. Los beneficios potenciales justifican mayor investigación al respecto. Este artículo resume la evidencia científica disponible con respecto a la revitalización de dientes inmaduros y maduros necróticos, sus fundamentos biológicos, los resultados esperados y limitaciones, así como el protocolo clínico.

ABSTRACT: Nowadays, the treatment of immature necrotic teeth is an important clinical challenge. The absence of apex closure and low thickness of the dentin walls, make endodontic treatment unpredictable and difficult. Traditionally, these teeth have been treated with apexification and obturation of the root canal. As a result of this treatment, the tooth remains devitalized and with fragile and short dentin walls, which compromises its prognosis. Regenerative endodontics, on the other hand, seeks to revitalize the tooth and induce root maturation, and is based on the use of mesenchymal stem cells present in the periapical tissues, growth factors present in the dentin and a scaffold that allows growth of new tissue in the root ca- nal. The clinical results are encouraging, since generally, there is root maturation and revascularization of the tooth. However, the newly formed tissue is reparative tissue and with the exception of some studies, no regeneration of dentin and pulp has been reported. Regenerative endodontics emerged to treat necrotic immature teeth. However, recently, preliminary studies have applied regenerative endodontics in mature necrotic teeth, in adult patients. Preliminary results are positive and are similar to those of immature teeth. Nevertheless, research regarding the revitalization of mature teeth is in the early stages and requires further evidence before being systematically administered as therapy in adult patients. However, the potential benefits justify further research in this regard. This article summarizes the available scientific evidence regarding the revitalization of immature and mature necrotic teeth, their biological basis, the expected results and limitations, as well as the clinical protocols for each case.

Association of polypropylene scaffolds and mesenchymal stem cells for use in tissue engineering / Associação de arcabouço de polipropileno e células tronco mesenquimais para uso em engenharia de tecido

Tissue engineering replaces injured tissues by manipulating cells, making scaffolds, and using molecules that stimulate the tissue. Mesenchymal stem cells (MSCs) are good candidates for tissue engineering, as this is one of the cell types which are recruited to repair injured tissues. Scaffolds are structural devices that allow cell fixation and migration, with polypropylene meshes being an example. This study aims to evaluate the culture of adipose tissue-derived mesenchymal stem cells (ADSCs), isolated from C57Bl/6 GFP + mice, in two types of polypropylene meshes (macroporous and microporous) in conventional culture plates and plates coated with methacrylate, over a period of fifteen days. The objective was to obtain the best interaction protocol between the mesh and the cells. The choice of the best method was based on adherence, maintenance of adherence and viability during culture. The amount of ADSCs adhering was checked daily by counting in a Neubauer Chamber and by using a growth curve performed with the MTT assay. The ADSCs adhering to the meshes were visualized with DAPI, panotic, hematoxylin and eosin, immunohistochemistry (integrin), and immunofluorescence (actin). ADSCs adhere to all forms of culture and to the two types of polypropylene mesh. ADSCs adhered more to the microporous mesh, within the seven day period of culture and in the plates without methacrylate. Thus, polypropylene meshes offer a good scaffold for ADSCs to adhere to.

A engenharia de tecidos substitui tecidos danificados com a manipulação de células, confecção de arcabouços e a utilização de moléculas que estimulem o tecido. As células-tronco mesenquimais (MSCs) são boas candidatas para engenharia de tecido, pois são um dos tipos celulares recrutadas para a reparação de tecidos lesionados. O arcabouço deve ser um dispositivo estrutural que forneça uma estrutura para o crescimento e a diferenciação celular no sítio, sendo a tela de polipropileno um exemplo. O objetivo deste estudo foi avaliar o cultivo de células-tronco mesenquimais de tecido de adiposo (ADSCs), isoladas de camundongos C57Bl/6 GFP+, em dois tipos de telas de polipropileno (macroporosa e microporosa) em placas de cultura convencionais e revestidas com metacrilato, durante quinze dias, para obter o melhor protocolo de interação entre a tela e as células. A escolha do melhor método foi baseada na adesão, manutenção da adesão e viabilidade durante cultivo. A quantidade de ADSCs aderidas foi verificada diariamente em contagem em Câmara de Neubauer e através de uma curva de crescimento realizada através de ensaio de MTT. As ADSCs aderidas nas telas foram visualizadas com a marcação de DAPI, panótico, hematoxilina e eosina, imumo-histoquímica (integrina) e imunofluorescência (actina). Nas duas formas de cultivo e nos dois tipos de telas de polipropileno houve aderência das ADSCs. Houve maior aderência na tela microporosa, no período de sete dias de cultivo e em placas sem metacrilato. Conclui-se que a tela de polipropileno oferece um bom arcabouço para as ADSCs se aderirem.

Enhanced osseointegration of porous titanium scaffold implanted with preload: an experiment study in rabbits / Osteointegración mejorada de malla de titanio poroso implantada con precarga: un estudio experimental en conejos

Porous titanium alloy scaffold was widely used in treating bone defect caused by traumatic injury and osteomyelitis, which was incapable of self-healing. The implantation of scaffold produced stress shielding thereby forming osteolysis. The objective of this study was to analysis trabecular morphological features of osseointegrated bone. 14 New Zealand rabbits were divided into two groups, surgery group and healthy control group. 7 rabbits in surgery group were selected to perform 3D printed porous titanium alloy scaffold implantation surgery with preload at the defect of femoral condyle for osseointegration. The other 7 rabbits in control group were feed free. After 90 days healing, femoral condyles were extracted to perform micro-CT scanning with hydroxyapatite calibration phantom. Mean bone mineral density (BMD), bone volume fraction (BV/TV), BS/TV (bone surface area ratio), Tb.Th (thickness of trabeculae), Tb.N (number of trabeculae), Tb.Sp (trabecular separation) and DA (degree of anisotropy) were calculated from micro-CT images. The results revealed that osseointegration inside and at the surface of scaffolds worked well from grey values of micro-CT images. After 12 weeks healing, mean bone mineral densities (BMD) in surgery group and healthy control group were calculated as 800±20mg/cm3 and 980±90mg/cm3, respectively. This revealed that the strength of trabeculae in surgery group might lower than that in the healthy group. Trabecular morphological parameters test showed that trabecular morphological parameters at the surface of scaffolds in the surgery group deteriorated significantly. It was found from micro-CT images that ingrowth bone was filled with pores of scaffold. Overall, the effect of osseointegration was promoted through the change of mechanical micro-environment in the scaffold region. Overall, preload could improve osseointegration effect in the long-term after surgery. However, the trabecular morphology in the surgery group was deteriorated, which might bring secondary fracture risk again.

La malla de aleación de titanio poroso se usó ampliamente en el tratamiento de defectos óseos causados por lesiones traumáticas y osteomielitis. El implante de la malla generó una protección contra el estrés, formando así osteolisis. El objetivo de este estudio fue analizar las características morfológicas trabeculares del hueso osteointegrado. Se dividieron 14 conejos (Neozelandeses) en dos grupos, grupo cirugía y grupo control saludable. Se seleccionaron 7 conejos en el grupo de cirugía para realizar una implantación de mallas de aleación de titanio poroso, impresas en 3D con precarga en el defecto del cóndilo femoral para la osteointegración. Los 7 conejos restantes del grupo control se mantuvieron sin alimentación. Después de 90 días de curación, se extrajeron los cóndilos femorales para realizar una exploración por micro-CT con un espectro de calibración de hidroxiapatita. Se calcularon a partir de imágenes de micro-CTDensidad mineral ósea media (DMO), fracción de volumen óseo (BV / TV), BS / TV (relación de área de superficie ósea), Tb.Th (espesor de trabéculas), Tb.N (número de trabéculas), Tb.Sp (trabecular separación) y DA (grado de anisotropía). Los resultados revelaron que la osteointegración dentro y en la superficie de los andamios funcionó bien a partir de los valores grises de las imágenes de micro-CT. Después de 12 semanas de curación, las densidades medias de minerales óseos (DMO) en el grupo cirugía y en el grupo control sano se calcularon como 800 ± 20 mg/cm3 y 980 ± 90 mg/cm3, respectivamente. Esto reveló que la fuerza de las trabéculas en el grupo de cirugía podría ser menor que la del grupo sano. La prueba de parámetros morfológicos trabeculares mostró que en el grupo de cirugía, la superficie de las mallas, se deterioraron significativamente. Se descubrió a partir de imágenes de microCT que el hueso en crecimiento estaba lleno de poros de andamio. En general, el efecto de la osteointegración se promovió mediante el cambio del microambiente mecánico en la región de la malla. En general, la precarga podría mejorar el efecto de osteointegración a largo plazo después de la cirugía. Sin embargo, la morfología trabecular en el grupo de cirugía se deterioró, lo que podría traer un nuevo riesgo de fractura secundaria.

Magnesium versus poly-L-lactic acid bioresorbable scaffolds: in vivo optical coherence tomography comparison of mechanical performance / Andamiajes bioresorbibles de magnesio versus poliméricos: comparación in vivo de su comportamiento mecánico por OCT

Abstract Background: Different mechanical properties have been suggested for metallic bioresorbable vascular scaffolds (BVS) in comparison to polymeric BVS. We aim to evaluate the acute mechanical performance of Magmaris® scaffold in comparison to Absorb®. Materials and Methods: Two groups of 10 coronary lesions treated with Magmaris® and Absorb® 1.1 (20584 vs. 21016 struts) were compared. In all cases, optical coherence tomographic (OCT) images were acquired after scaffold deployment. Baseline clinical, angiographic, and procedural characteristics were compared, including OCT evaluations. Results: No baseline clinical or angiographic significant differences were found between groups. The most common indication for revascularization was effort angina (60% vs. 70% p = 0.45) with no ST-elevation myocardial infarction (MI) cases. Main target artery was left anterior descending, with a mean vessel diameter of 3.46 ± 0.23 in Absorb® and 3.52 ± 0.19mm in Magmaris® groups (p = 0.56). All cases underwent pre- and post-dilatation with a procedural success rate of 100%. OCT analyses showed larger scaffold and vessel diameters in Magmaris® group: 3.11 ± 0.38 mm versus 3.07 ± 0.36 mm, p = 0.03 and 4.12 ± 0.51 mm versus 4.04 ± 0.46 mm, p = 0.04. Despite the application of slightly higher postdilatation pressures to Magmaris® devices (18.01 ± 2.15 vs. 17.20 ± 3.80 atm, p = 0.05), significantly lower percentages of disrupted and malapposed struts were identified within Magmaris® scaffolds (0.15% vs. 0.27%, p = 0.03 and 1.06% vs. 1.46% p = 0.01). No cardiac death, target vessel-related MI, or clinically driven target lesion revascularization was reported in a 30-day follow-up. Conclusion: Mechanical properties of Magmaris® scaffold allow achieving larger vessel and scaffold diameters in a safe manner, with lower rates of malapposition and scaffold disruption.

Resumen Introducción: Se ha sugerido la presencia de un distinto comportamiento mecánico entre los dos grupos principales de dispositivos bioresorbibles: metálicos y poliméricos. En este estudio evaluamos el comportamiento mecánico agudo del andamiaje bioresorbible metálico Magmaris® frente al del polimérico Absorb®. Métodos: Se compararon dos grupos de 10 lesiones coronarias tratadas con Magmaris® y Absorb® 1.1 (20584 vs. 21016 struts). En todos los casos se realizó estudio postimplante del dispositivo mediante tomografia de coherencia óptica (OCT). Se compararon las características basales clínicas y angiográficas, así como aspectos del procedimiento (incluídos los estudios de OCT) entre ambos grupos. Resultados: No se encontraron diferencias clínicas o angiográficas estadísticamente significativas entre ambos grupos. La indicación más frecuente de revascularización coronaria fué la presencia de angina de esfuerzo (60% vs. 70% p = 0.45), sin incluirse casos de IAMCEST. La arteria descendente anterior fué el principal vaso diana, con un diámetro medio de 3.46 ± 0.23 mm en el grupo de Absorb® y de 3.52 ± 0.19mm en el grupo de Magmaris® (p = 0.56). En todos los casos se realizó pre y postdilatación, con una tasa de éxito del procedimiento del 100%. Los estudios mediante OCT demostraron un mayor diámetro de stent y del vaso en el grupo de Magmaris®: 3.11 ± 0.38mm versus 3.07 ± 0.36 mm, p = 0.03 y 4.12 ± 0.51mm versus 4.04 ± 0.46mm, p = 0.04. A pesar de someter a los dispositivos Magmaris® a presiones de postdilatación ligeramente superiores (18.01 ± 2.15 vs. 17.20 ± 3.80 atm, p = 0.05), se identificó un menor porcentaje estadísticamente significativo de struts rotos o malapuestos en dicho grupo (0.15% vs. 0.27 %, p = 0.03 y 1.06 % vs. 1.46% p = 0.01). En un seguimiento a 30 días no se registraron eventos mayores: muerte cardíaca, IM relacionado con vaso diana o TLR. Conclusión: Las propiedades mecánicas del scaffold metálico bioresorbible Magmaris® permiten alcanzar mayores diámetros de stent y vaso de forma segura tras su implante, con una baja tasa de malaposición y disrupción.

Importancia de la angiogénesis en el diseño de scaffolds para ingeniería de tejido óseo / Importance of angiogenesis in the design of scaffolds for bone tissue engineering

Hematoma, inflamación, angiogénesis y osteogénesis son distintas etapas que se superponen durante el proceso de reparación de una fractura ósea. Durante las primeras etapas se liberan distintos factores de crecimiento quimioatractantes que producen el reclutamiento de diversas células para generar la formación de un hueso funcional con su respectiva vasculatura. Debido a la importancia que posee la angiogénesis en el desarrollo de una adecuada red vascular, tanto para la formación ósea como en su reparación, en los últimos años los especialistas en ingeniería de tejido óseo han estudiado la manera de fomentar tanto la osteogénesis como la angiogénesis durante la reparación ósea. En este trabajo de revisión, se recopilan y discuten los principales conceptos sobre distintas estrategias a fin de lograr un implante sintético con funcionalidad dual promoviendo los procesos que garanticen la angiogénesis y la osteogénesis en forma acoplada utilizando distintos tipos de scaffolds y sistemas de liberación de drogas osteoinductoras y angioinductoras. La liberación dual de factores osteoinductores y angioinductores debe producirse en forma témporo-espacial controlada para garantizar los efectos deseados sin producir efectos adversos como tumores o hueso ectópico. Se deben tener en cuenta varios factores como el tipo y la arquitectura de hueso, tipo de daño, edad, sexo y condiciones patológicas del paciente. En cuanto a los materiales se debe considerar el tipo de material para usar como scaffold, los factores inductores seleccionados, su combinación y sistemas de liberación. El avance en estos estudios hará que la Ingeniería de Tejido Óseo sea una alternativa terapéutica en el futuro. (AU)

Hematoma, inflammation, angiogenesis, and osteogenesis are different stages that overlap during the healing process of a bone fracture. During the first stages, different chemoattractant growth factors are released which produce the recruitment of various cells that will induce the formation of a functional bone with its respective vasculature. Due to the importance of angiogenesis for the development of an adequate vascular network in both bone formation and repair, in recent years specialists in bone tissue engineering have studied how to promote both osteogenesis and angiogenesis during bone repair. In this review, the main concepts on different strategies developed to achieve a synthetic implant with dual functionality, promoting processes that guarantee angiogenesis and osteogenesis in a coupled way using different types of scaffolds and osteo-drug delivery systems and angioinductors, are collected and discussed. The dual release for osteoinductive and angioinductive factors must ensure the release of them in a controlled time-space manner to guarantee the desired effects without producing adverse effects such as tumors or ectopic bone. Several factors must be taken into account, such as bone type and architecture, type of damage to be repaired, age, sex, and pathological conditions of the patient. Regarding the materials, the type of material to be used as scaffolds, selected inducing factors and drug release system must be considered. Advances in these studies will make Bone Tissue Engineering a therapeutic alternative in the future. (AU)

Bioactive Fibrin Scaffolds for Use in Musculoskeletal Regenerative Medicine

Abstract Autologous fibrin matrices derived from the Leukocyte and Platelet Rich Plasma (L-PRP) and Leukocyte and Platelet Rich Fibrin (L-PRF) techniques present great potential to act as a bioactive scaffold in regenerative medicine, contributing to the maintenance of cell viability, proliferation stimulus and differentiation. In contrast, there are few studies that characterize the bioactive potential of these fibrin scaffolds by considering the process of production. The objective of this work was to characterize the intrinsic potential of maintaining cell viability of different fibrin scaffolds containing platelets and leukocytes. In order to achieve that, blood samples from a volunteer were collected and processed to obtain fibrin clots using the suggested techniques. To characterize the potential for in vitro viability, mesenchymal stem cells from human infrapatellar fat were used. The scaffolds were cellularized (1x105 cells/scaffolds) and maintained for 5 and 10 days under culture conditions with Dulbecco's Modified Eagle Medium, without addition of fetal bovine serum, and subsequently subjected to analyses by Fourrier transform infra-red spectroscopy, circular dichroism and fluorescence microscopy. The results demonstrated distinct intrinsic potential viability between the scaffolds, and L-PRP was responsible for promoting higher levels of viability in both periods of analysis. No viable cells were identified in the fibrin matrix used as controls. These results allow us to conclude that both fibrin substrates have presented intrinsic potential for maintaining cell viability, with superior potential exhibited by L-PRP scaffold, and represent promising alternatives for use as bioactive supports in musculoskeletal regenerative medicine.

Uso da pele de tilápia do Nilo em medicina regenerativa: Status atual e perspectivas futuras / Nile tilapia fish skin use in regenerative medicine: Current status and future perspectives

OBJETIVO: O presente estudo tem como objetivo apresentar a trajetória do desenvolvimento da pele de tilápia como curativo biológico. MÉTODO: Descrição do processo de desenvolvimento da pele de tilápia como curativo biológico, desde a concepção da ideia para utilização no tratamento de queimaduras, até as perspectivas de sua utilização como scaffold em múltiplas especialidades médicas. RESULTADOS: Com o objetivo de oferecer uma alternativa segura, eficaz e de baixo custo para o tratamento de queimaduras, pesquisadores brasileiros desenvolveram um curativo biológico derivado da pele de tilápia. CONCLUSÃO: A pele de tilápia vem demonstrando um notável potencial como curativo biológico no tratamento de queimaduras e na medicina regenerativa.

OBJECTIVE: The present study aims to present the development trajectory of the tilapia skin as a biological dressing. METHODS: To describe the development process of tilapia skin as biological dressing, from the conception to the burn wounds treatment to the perspectives of its use as a scaffold in multiple medical specialties. RESULTS: In order to provide a safe, effective and low-cost alternative for the treatment of burn injuries, Brazilian researchers have recently developed a biological dressing derived from tilapia skin. CONCLUSION: The tilapia skin have been demonstrating the remarkable potential as a biological dressing in burn injuries treatment and in regenerative medicine.

VPA/PLGA microfibers produced by coaxial electrospinning for the treatment of central nervous system injury

The central nervous system shows limited regenerative capacity after injury. Spinal cord injury (SCI) is a devastating traumatic injury resulting in loss of sensory, motor, and autonomic function distal from the level of injury. An appropriate combination of biomaterials and bioactive substances is currently thought to be a promising approach to treat this condition. Systemic administration of valproic acid (VPA) has been previously shown to promote functional recovery in animal models of SCI. In this study, VPA was encapsulated in poly(lactic-co-glycolic acid) (PLGA) microfibers by the coaxial electrospinning technique. Fibers showed continuous and cylindrical morphology, randomly oriented fibers, and compatible morphological and mechanical characteristics for application in SCI. Drug-release analysis indicated a rapid release of VPA during the first day of the in vitro test. The coaxial fibers containing VPA supported adhesion, viability, and proliferation of PC12 cells. In addition, the VPA/PLGA microfibers induced the reduction of PC12 cell viability, as has already been described in the literature. The biomaterials were implanted in rats after SCI. The groups that received the implants did not show increased functional recovery or tissue regeneration compared to the control. These results indicated the cytocompatibility of the VPA/PLGA core-shell microfibers and that it may be a promising approach to treat SCI when combined with other strategies.