Polydioxanone-Based Membranes for Bone Regeneration.

Resorbable synthetic and natural polymer-based membranes have been extensively studied for guided tissue regeneration. Alloplastic biomaterials are often used for tissue regeneration due to their lower immunoreactivity when compared with allogeneic and xenogeneic materials. Plenum® Guide is a synthetic membrane material based on polydioxanone (PDO), whose surface morphology closely mimics the extracellular matrix. In this study, Plenum® Guide was compared with collagen membranes as a barrier material for bone-tissue regeneration in terms of acute and subchronic systemic toxicity. Moreover, characterizations such as morphology, thermal analysis (Tm = 107.35 °C and crystallinity degree = 52.86 ± 2.97 %, final product), swelling (thickness: 0.25 mm ≅ 436% and 0.5 mm ≅ 425% within 24 h), and mechanical tests (E = 30.1 ± 6.25 MPa; σ = 3.92 ± 0.28 MPa; ε = 287.96 ± 34.68%, final product) were performed. The in vivo results revealed that the PDO membranes induced a slightly higher quantity of newly formed bone tissue than the control group (score: treated group = 15, control group = 13) without detectable systemic toxicity (clinical signs and evaluation of the membranes after necropsy did not result in differences between groups, i.e., non-reaction -> tissue-reaction index = 1.3), showing that these synthetic membranes have the essential characteristics for an effective tissue regeneration. Human adipose-derived stem cells (hASCs) were seeded on PDO membranes; results demonstrated efficient cell migration, adhesion, spread, and proliferation, such that there was a slightly better hASC osteogenic differentiation on PDO than on collagen membranes. Hence, Plenum® Guide membranes are a safe and efficient alternative for resorbable membranes for tissue regeneration.

Adipose Tissue-Derived Stem Cells: The Biologic Basis and Future Directions for Tissue Engineering.

Mesenchymal stem cells (MSCs) have been isolated from a variety of tissues using different methods. Active research have confirmed that the most accessible site to collect them is the adipose tissue; which has a significantly higher concentration of MSCs. Moreover; harvesting from adipose tissue is less invasive; there are no ethical limitations and a lower risk of severe complications. These adipose-derived stem cells (ASCs) are also able to increase at higher rates and showing telomerase activity, which acts by maintaining the DNA stability during cell divisions. Adipose-derived stem cells secret molecules that show important function in other cells vitality and mechanisms associated with the immune system, central nervous system, the heart and several muscles. They release cytokines involved in pro/anti-inflammatory, angiogenic and hematopoietic processes. Adipose-derived stem cells also have immunosuppressive properties and have been reported to be "immune privileged" since they show negative or low expression of human leukocyte antigens. Translational medicine and basic research projects can take advantage of bioprinting. This technology allows precise control for both scaffolds and cells. The properties of cell adhesion, migration, maturation, proliferation, mimicry of cell microenvironment, and differentiation should be promoted by the printed biomaterial used in tissue engineering. Self-renewal and potency are presented by MSCs, which implies in an open-source for 3D bioprinting and regenerative medicine. Considering these features and necessities, ASCs can be applied in the designing of tissue engineering products. Understanding the heterogeneity of ASCs and optimizing their properties can contribute to making the best therapeutic use of these cells and opening new paths to make tissue engineering even more useful.

AUTOLOGOUS CHONDROCYTE IMPLANTATION IN BRAZIL.

OBJECTIVE: To describe the first series of cases of autologous chondrocyte implantation (ACI) in collagen membrane performed in Brazil. METHODS: ACI was performed in 12 knees of 11 patients, aged 32.1 ± 10.9 years, with 5.3 ± 2.6 cm2 full-thickness knee cartilage lesions, with a six-month minimum follow-up. Two surgical procedures were performed: arthroscopic cartilage biopsy for isolation and expansion of chondrocytes, which were seeded onto collagen membrane and implanted in the lesion site; the characterization of cultured cells and implant was performed using immunofluorescence for type II collagen (COL2) for cell viability and electron microscopy of the implant. Clinical safety, KOOS and IKDC scores and magnetic resonance imaging were evaluated. We used repeated-measures ANOVA and post-hoc comparisons at α = 5%. RESULTS: COL2 was identified in the cellular cytoplasm, cell viability was higher than 95% and adequate distribution and cell adhesion were found in the membrane. The median follow-up was 10.9 months (7 to 19). We had two cases of arthrofibrosis, one of graft hypertrophy and one of superficial infection as complications, but none compromising clinical improvement. KOOS and IKDC ranged from 71.2 ± 11.44 and 50.72 ± 14.10, in preoperative period, to 85.0 ± 4.4 and 70.5 ± 8.0, at 6 months (p = 0.007 and 0.005). MRI showed regenerated tissue compatible with hyaline cartilage. CONCLUSION: ACI in collagen membrane was feasible and safe in a short-term follow-up, presenting regenerated formation visualized by magnetic resonance imaging and improved clinical function. Level of evidence IV, Case series.

OBJETIVO: Descrever a primeira série de casos de transplante autólogo de condrócitos (TAC) em membrana de colágeno realizada no Brasil. MÉTODOS: Doze joelhos de onze pacientes, com idade de 32,1 ± 10,9 anos, com lesões de cartilagem de espessura total do joelho de tamanho de 5,3 ± 2,6 cm 2 foram submetidos ao TAC, com seguimento mínimo de seis meses. Realizamos dois procedimentos cirúrgicos: biópsia artroscópica de cartilagem para isolamento e expansão de condrócitos, que foram semeados em uma membrana de colágeno implantada no leito da lesão. Foi realizada caracterização com imunofluorescência para colágeno tipo II (COL2) de células cultivadas e implantes, viabilidade celular e microscopia eletrônica no implante. Foram avaliados a segurança clínica, os escores funcionais KOOS e IKDC e a ressonância magnética. Utilizamos teste ANOVA para medidas repetidas, com comparações post-hoc, α = 5%. RESULTADOS: COL2 foi identificado no citoplasma da célula, viabilidade celular foi superior a 95% e houve distribuição adequada e adesão celular na membrana. O seguimento mediano foi de 10,9 meses (7 a 19). Como complicações, ocorreram dois casos de artrofibrose, um de hipertrofia do enxerto e um de infecção superficial, nenhum deles havendo comprometimento da melhora clínica. Escalas KOOS e IKDC passaram de 71,2 ± 11,44 e 50,72 ± 14,10, no pré-operatório, para 85,0 ± 4,4 e 70,5 ± 8,0, aos 6 meses (p = 0,007 e 0,005). Ressonância magnética mostrou tecido regenerado compatível com cartilagem hialina. CONCLUSÃO: TAC em membrana de colágeno foi viável e seguro em seguimento de curto prazo, apresentando formação de regenerado visualizado através de imagens de ressonância magnética e melhora de função clínica. Nível de evidência IV, Série de casos.

IMPLEMENTAÇÃO DO TRANSPLANTE AUTÓLOGO DE CONDRÓCItOS NO BRASIL / Autologous chondrocyte implantation in brazil

ABSTRACT Objective: To describe the first series of cases of autologous chondrocyte implantation (ACI) in collagen membrane performed in Brazil. Methods: ACI was performed in 12 knees of 11 patients, aged 32.1 ± 10.9 years, with 5.3 ± 2.6 cm2 full-thickness knee cartilage lesions, with a six-month minimum follow-up. Two surgical procedures were performed: arthroscopic cartilage biopsy for isolation and expansion of chondrocytes, which were seeded onto collagen membrane and implanted in the lesion site; the characterization of cultured cells and implant was performed using immunofluorescence for type II collagen (COL2) for cell viability and electron microscopy of the implant. Clinical safety, KOOS and IKDC scores and magnetic resonance imaging were evaluated. We used repeated-measures ANOVA and post-hoc comparisons at α = 5%. Results: COL2 was identified in the cellular cytoplasm, cell viability was higher than 95% and adequate distribution and cell adhesion were found in the membrane. The median follow-up was 10.9 months (7 to 19). We had two cases of arthrofibrosis, one of graft hypertrophy and one of superficial infection as complications, but none compromising clinical improvement. KOOS and IKDC ranged from 71.2 ± 11.44 and 50.72 ± 14.10, in preoperative period, to 85.0 ± 4.4 and 70.5 ± 8.0, at 6 months (p = 0.007 and 0.005). MRI showed regenerated tissue compatible with hyaline cartilage. Conclusion: ACI in collagen membrane was feasible and safe in a short-term follow-up, presenting regenerated formation visualized by magnetic resonance imaging and improved clinical function. Level of evidence IV, Case series.

RESUMO Objetivo: Descrever a primeira série de casos de transplante autólogo de condrócitos (TAC) em membrana de colágeno realizada no Brasil. Métodos: Doze joelhos de onze pacientes, com idade de 32,1 ± 10,9 anos, com lesões de cartilagem de espessura total do joelho de tamanho de 5,3 ± 2,6 cm 2 foram submetidos ao TAC, com seguimento mínimo de seis meses. Realizamos dois procedimentos cirúrgicos: biópsia artroscópica de cartilagem para isolamento e expansão de condrócitos, que foram semeados em uma membrana de colágeno implantada no leito da lesão. Foi realizada caracterização com imunofluorescência para colágeno tipo II (COL2) de células cultivadas e implantes, viabilidade celular e microscopia eletrônica no implante. Foram avaliados a segurança clínica, os escores funcionais KOOS e IKDC e a ressonância magnética. Utilizamos teste ANOVA para medidas repetidas, com comparações post-hoc, α = 5%. Resultados: COL2 foi identificado no citoplasma da célula, viabilidade celular foi superior a 95% e houve distribuição adequada e adesão celular na membrana. O seguimento mediano foi de 10,9 meses (7 a 19). Como complicações, ocorreram dois casos de artrofibrose, um de hipertrofia do enxerto e um de infecção superficial, nenhum deles havendo comprometimento da melhora clínica. Escalas KOOS e IKDC passaram de 71,2 ± 11,44 e 50,72 ± 14,10, no pré-operatório, para 85,0 ± 4,4 e 70,5 ± 8,0, aos 6 meses (p = 0,007 e 0,005). Ressonância magnética mostrou tecido regenerado compatível com cartilagem hialina. Conclusão: TAC em membrana de colágeno foi viável e seguro em seguimento de curto prazo, apresentando formação de regenerado visualizado através de imagens de ressonância magnética e melhora de função clínica. Nível de evidência IV, Série de casos.

A Reliable Stem Cell Carrier: An Experimental Study in Wistar Rats.

INTRODUCTION: Treatments based on cell biology need reliable and precise carriers for reaching the desired targets. For that reason, a PDO-based cell carrier was idealized, with the purpose of carrying stem cells to distant sites at room temperature. MATERIALS AND METHODS: Three modalities of the same carrier were evaluated: one containing undifferentiated human dental pulp stem cells (DPSCs); one loaded with stem cells induced to neurogenic differentiation (DPSCNs); and one without cells (Blank). The carriers were implanted in sciatic nerve gaps in 48 Wistar rats that were divided in three groups. Two other rats were included in a SHAM control group. Immunohistochemical, histological and clinical analyses were performed in two, four, six and eight weeks of time. RESULTS: Efficacy of human stem cell transportation at room temperature to rats was attested. Moreover, it was possible to confirm that those cells show tropism for inflamed environments and are also prone to induction of neurogenesis in the first two weeks, vanishing after that period. CONCLUSION: Clinical evaluation of the animals' gait recovery shows a promising perspective of success with the inclusion of stem cell-loaded PDO tubes in nerve gaps, which may be positively compared to previously published studies. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors - www.springer.com/00266.

Scaling-up of dental pulp stem cells isolated from multiple niches.

Dental pulp (DP) can be extracted from child's primary teeth (deciduous), whose loss occurs spontaneously by about 5 to 12 years. Thus, DP presents an easy accessible source of stem cells without ethical concerns. Substantial quantities of stem cells of an excellent quality and at early (2-5) passages are necessary for clinical use, which currently is a problem for use of adult stem cells. Herein, DPs were cultured generating stem cells at least during six months through multiple mechanical transfers into a new culture dish every 3-4 days. We compared stem cells isolated from the same DP before (early population, EP) and six months after several mechanical transfers (late population, LP). No changes, in both EP and LP, were observed in morphology, expression of stem cells markers (nestin, vimentin, fibronectin, SH2, SH3 and Oct3/4), chondrogenic and myogenic differentiation potential, even after cryopreservation. Six hours after DP extraction and in vitro plating, rare 5-bromo-2'-deoxyuridine (BrdU) positive cells were observed in pulp central part. After 72 hours, BrdU positive cells increased in number and were found in DP periphery, thus originating a multicellular population of stem cells of high purity. Multiple stem cell niches were identified in different zones of DP, because abundant expression of nestin, vimentin and Oct3/4 proteins was observed, while STRO-1 protein localization was restricted to perivascular niche. Our finding is of importance for the future of stem cell therapies, providing scaling-up of stem cells at early passages with minimum risk of losing their "stemness".

Mesenchymal progenitor cells from canine fetal tissues: yolk sac, liver, and bone marrow.

During fetal development, mesenchymal progenitor (MP) cells are co-localized in major hematopoietic territories, such as yolk sac (YS), bone marrow (BM), liver (LV), and others. Studies using mouse and human MP cells isolated from fetus have shown that these cells are very similar but not identical to adult mesenchymal stem cells (MSC). Their differentiation potential is usually restricted to production of highly committed osteogenic and chondrogenic precursors. Such properties of fetal MP cells can be very useful for tissue regeneration, when a great number of committed precursors are required. The objectives of this study were to isolate and characterize MP cells from canine YS, BM, and LV in early and late stages of fetal development. Gestational stage was identified, and cell culture conditions were evaluated for efficient isolation of canine MP cells. All canine fetal MP cells expressed vimentin, nestin, and CD44 proteins. Cytokeratin 18 expression was observed in BM- and LV-MP cells, and vascular endothelial (VE)-cadherin expression was observed only in YS-MP cells. A small number of MP cells (5%) from LV and YS expressed Oct3/4 protein. The differentiation potential of canine fetal MP cells varied significantly: YS- and BM-MP cells differentiated into bone and cartilage, whereas LV-MP cells differentiation was limited to osteogenic fate. None of the canine fetal MP cells were able to differentiate into adipose cells. Our data suggest that canine fetal MP cells are an appropriate in vitro model to study MP biology from hematopoietic territories and they are a source of committed osteogenic and chondrogenic precursors for regenerative medicine.

Feeder-free derivation of induced pluripotent stem cells from human immature dental pulp stem cells.

Induced pluripotent stem cells (iPSCs) can be created by forcing expression of certain genes in fibroblasts or other somatic cell types, reversing them to a pluripotent state similar to that of embryonic stem cells (ESC). Here, we used human immature dental pulp stem cells (hIDPSCs) as an alternative source for creating iPSC. hIDPSCs can be easily isolated from accessible tissue of young and adult patients. hIDPSCs possess a fibroblast-like morphology, retaining characteristics of adult multipotent stem cells. Reprogramming of hIDPSCs was fast, producing primary hIDPSC-iPSC colonies even under feeder-free conditions. hIDPSCs acquired ESC-like morphology, expressed pluripotent markers, possessed stable, normal karyotypes, and demonstrated the ability to differentiated in vitro and in vivo. Our data demonstrate that hIDPSCs-iPSCs offer an advantageous cell system for future cell therapy and basic studies, particularly as a model for pediatric developmental disorders.

Long-Term Culture of Mouse EmbryonicStem Cell-Derived Adherent Neurospheres and Functional Neurons

Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neuronsfunctional properties and features, have been developed. Most of these protocols are short lasting, which,therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describehere a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). The AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. The expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during3 months under several splitting...

Long-term culture of mouse embryonic stem cell-derived adherent neurospheres and functional neurons.

Innumerous protocols, using the mouse embryonic stem (ES) cells as model for in vitro study of neurons functional properties and features, have been developed. Most of these protocols are short lasting, which, therefore, does not allow a careful analysis of the neurons maturation, aging, and death processes. We describe here a novel and efficient long-lasting protocol for in vitro ES cells differentiation into neuronal cells. It consists of obtaining embryoid bodies, followed by induction of neuronal differentiation with retinoic acid of nonadherent embryoid bodies (three-dimensional model), which further allows their adherence and formation of adherent neurospheres (AN, bi-dimensional model). The AN can be maintained for at least 12 weeks in culture under repetitive mechanical splitting, providing a constant microenvironment (in vitro niche) for the neuronal progenitor cells avoiding mechanical dissociation of AN. The expression of neuron-specific proteins, such as nestin, sox1, beta III-tubulin, microtubule-associated protein 2, neurofilament medium protein, Tau, neuronal nuclei marker, gamma-aminobutyric acid, and 5-hydroxytryptamine, were confirmed in these cells maintained during 3 months under several splitting. Additionally, expression pattern of microtubule-associated proteins, such as lissencephaly (Lis1) and nuclear distribution element-like (Ndel1), which were shown to be essential for differentiation and migration of neurons during embryogenesis, was also studied. As expected, both proteins were expressed in undifferentiated ES cells, AN, and nonrosette neurons, although presenting different spatial distribution in AN. In contrast to previous studies, using cultured neuronal cells derived from embryonic and adult tissues, only Ndel1 expression was observed in the centrosome region of early neuroblasts from AN. Mature neurons, obtained from ES cells in this work, display ionic channels and oscillations of membrane electrical potential typical of electrically excitable cells, which is a characteristic feature of the functional central nervous system (CNS) neurons. Taken together, our study demonstrated that AN are a long-term culture of neuronal cells that can be used to analyze the process of neuronal differentiation dynamics. Thus, the protocol described here provides a new experimental model for studying neurological diseases associated with neuronal differentiation during early development, as well as it represents a novel source of functional cells that can be used as tools for testing the effects of toxins and/or drugs on neuronal cells.

Corneal Reconstruction with Tissue-Engineered Cell Sheets Composed of Human Immature Dental Pulp Stem Cells

To determine the outcome of the use of a tissue-engineered cell sheet composed of human undifferentiated immature dental pulp stem cells (hIDPSC) for ocular surface reconstruction in an animal model of total limbal stem cell deficiency (LSCD). LSCD was induced by the application of 0.5 M NaOH to the right eye of rabbits for 25 seconds (mild chemical burn [MCB]) and for 45 seconds (severe chemical burn [SCB]). After 1 month, a superficial keratectomy was performed to remove the fibrovascular pannus that covered the animals' burned corneas. A tissue-engineered hIDPSC sheet was transplanted onto the corneal bed and then covered with deepithelialized human amniotic membrane (AM). In the respective control groups, the denuded cornea was covered with AM only. After 3 months, a detailed analysis of the rabbit eyes was performed with regard to clinical aspect, histology, electron microscopy, and immunohistochemistry. Corneal transparency of the rabbit eyes that underwent hIDPSC transplantation was improved throughout the follow-up, while the control corneas developed total conjunctivalization and opacification. Rabbits from the MCB group showed clearer corneas with less neovascularization. The clinical data were confirmed by histologic analysis that showed healthy uniform corneal epithelium, especially in the MCB group. The presence of hIDPSC was detected using an anti-hIDPSC antibody. The corneal tissue also showed positive immunostaining with anti-human antibodies. In the control corneas, none of these antigens were detected. Overall, these data showed that transplantation of a tissue-engineered hIDPSC sheet was successful for the reconstruction of corneal epithelium in an animal model of LSCD.

Corneal reconstruction with tissue-engineered cell sheets composed of human immature dental pulp stem cells.

PURPOSE: To determine the outcome of the use of a tissue-engineered cell sheet composed of human undifferentiated immature dental pulp stem cells (hIDPSC) for ocular surface reconstruction in an animal model of total limbal stem cell deficiency (LSCD). METHODS: LSCD was induced by the application of 0.5 M NaOH to the right eye of rabbits for 25 seconds (mild chemical burn [MCB]) and for 45 seconds (severe chemical burn [SCB]). After 1 month, a superficial keratectomy was performed to remove the fibrovascular pannus that covered the animals' burned corneas. A tissue-engineered hIDPSC sheet was transplanted onto the corneal bed and then covered with deepithelialized human amniotic membrane (AM). In the respective control groups, the denuded cornea was covered with AM only. After 3 months, a detailed analysis of the rabbit eyes was performed with regard to clinical aspect, histology, electron microscopy, and immunohistochemistry. RESULTS: Corneal transparency of the rabbit eyes that underwent hIDPSC transplantation was improved throughout the follow-up, while the control corneas developed total conjunctivalization and opacification. Rabbits from the MCB group showed clearer corneas with less neovascularization. The clinical data were confirmed by histologic analysis that showed healthy uniform corneal epithelium, especially in the MCB group. The presence of hIDPSC was detected using an anti-hIDPSC antibody. The corneal tissue also showed positive immunostaining with anti-human antibodies. In the control corneas, none of these antigens were detected. CONCLUSIONS: Overall, these data showed that transplantation of a tissue-engineered hIDPSC sheet was successful for the reconstruction of corneal epithelium in an animal model of LSCD.