Pretreatment of Mesenchymal Stem Cells with Electrical Stimulation as a Strategy to Improve Bone Tissue Engineering Outcomes.

Electrical stimulation (EStim), whether used alone or in combination with bone tissue engineering (BTE) approaches, has been shown to promote bone healing. In our previous in vitro studies, mesenchymal stem cells (MSCs) were exposed to EStim and a sustained, long-lasting increase in osteogenic activity was observed. Based on these findings, we hypothesized that pretreating MSC with EStim, in 2D or 3D cultures, before using them to treat large bone defects would improve BTE treatments. Critical size femur defects were created in 120 Sprague-Dawley rats and treated with scaffold granules seeded with MSCs that were pre-exposed or not (control group) to EStim 1 h/day for 7 days in 2D (MSCs alone) or 3D culture (MSCs + scaffolds). Bone healing was assessed at 1, 4, and 8 weeks post-surgery. In all groups, the percentage of new bone increased, while fibrous tissue and CD68+ cell count decreased over time. However, these and other healing features, like mineral density, bending stiffness, the amount of new bone and cartilage, and the gene expression of osteogenic markers, did not significantly differ between groups. Based on these findings, it appears that the bone healing environment could counteract the long-term, pro-osteogenic effects of EStim seen in our in vitro studies. Thus, EStim seems to be more effective when administered directly and continuously at the defect site during bone healing, as indicated by our previous studies.

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro.

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Time course of traumatic neuroma development.

This study was designed to characterize morphologic stages during neuroma development post amputation with an eye toward developing better treatment strategies that intervene before neuromas are fully formed. Right forelimbs of 30 Sprague Dawley rats were amputated and limb stumps were collected at 3, 7, 28, 60 and 90 Days Post Amputation (DPA). Morphology of newly formed nerves and neuromas were assessed via general histology and neurofilament protein antibody staining. Analysis revealed six morphological characteristics during nerve and neuroma development; 1) normal nerve, 2) degenerating axons, 3) axonal sprouts, 4) unorganized bundles of axons, 5) unorganized axon growth into muscles, and 6) unorganized axon growth into fibrotic tissue (neuroma). At early stages (3 & 7 DPA) after amputation, normal nerves could be identified throughout the limb stump and small areas of axonal sprouts were present near the site of injury. Signs of degenerating axons were evident from 7 to 90 DPA. From day 28 on, variability of nerve characteristics with signs of unorganized axon growth into muscle and fibrotic tissue and neuroma formation became visible in multiple areas of stump tissue. These pathological features became more evident on days 60 and 90. At 90 DPA frank neuroma formation was present in all stump tissue. By following nerve regrowth and neuroma formation after amputation we were able to identify 6 separate histological stages of nerve regrowth and neuroma development. Axonal regrowth was observed as early as 3 DPA and signs of unorganized axonal growth and neuroma formation were evident by 28 DPA. Based on these observations we speculate that neuroma treatment and or prevention strategies might be more successful if targeted at the initial stages of development and not after 28 DPA.

Pretreating mesenchymal stem cells with electrical stimulation causes sustained long-lasting pro-osteogenic effects.

BACKGROUND: Electrical stimulation (ES) has a long history of successful use in the clinical treatment of refractory, non-healing bone fractures and has recently been proposed as an adjunct to bone tissue-engineering treatments to optimize their therapeutic potential. This idea emerged from ES's demonstrated positive effects on stem cell migration, proliferation, differentiation and adherence to scaffolds, all cell behaviors recognized to be advantageous in Bone Tissue Engineering (BTE). In previous in vitro experiments we demonstrated that direct current ES, administered daily, accelerates Mesenchymal Stem Cell (MSC) osteogenic differentiation. In the present study, we sought to define the optimal ES regimen for maximizing this pro-osteogenic effect. METHODS: Rat bone marrow-derived MSC were exposed to 100 mV/mm, 1 hr/day for three, seven, and 14 days, then osteogenic differentiation was assessed at Day 14 of culture by measuring collagen production, calcium deposition, alkaline phosphatase activity and osteogenic marker gene expression. RESULTS: We found that exposing MSC to ES for three days had minimal effect, while seven and 14 days resulted in increased osteogenic differentiation, as indicated by significant increases in collagen and calcium deposits, and expression of osteogenic marker genes Col1a1, Osteopontin, Osterix and Calmodulin. We also found that cells treated with ES for seven days, maintained this pro-osteogenic activity long (for at least seven days) after discontinuing ES exposure. DISCUSSION: This study showed that while three days of ES is insufficient to solicit pro-osteogenic effects, seven and 14 days significantly increases osteogenic differentiation. Importantly, we found that cells treated with ES for only seven days, maintained this pro-osteogenic activity long after discontinuing ES exposure. This sustained positive osteogenic effect is likely due to the enhanced expression of RunX2 and Calmodulin we observed. This prolonged positive osteogenic effect, long after discontinuing ES treatment, if incorporated into BTE treatment protocols, could potentially improve outcomes and in doing so help BTE achieve its full therapeutic potential.

Combining electrical stimulation and tissue engineering to treat large bone defects in a rat model.

Bone Tissue engineering (BTE) has recently been introduced as an alternative to conventional treatments for large non-healing bone defects. BTE approaches mimic autologous bone grafts, by combining cells, scaffold, and growth factors, and have the added benefit of being able to manipulate these constituents to optimize healing. Electrical stimulation (ES) has long been used to successfully treat non-healing fractures and has recently been shown to stimulate bone cells to migrate, proliferate, align, differentiate, and adhere to bio compatible scaffolds, all cell behaviors that could improve BTE treatment outcomes. With the above in mind we performed in vitro experiments and demonstrated that exposing Mesenchymal Stem Cells (MSC) + scaffold to ES for 3 weeks resulted in significant increases in osteogenic differentiation. Then in in vivo experiments, for the first time, we demonstrated that exposing BTE treated rat femur large defects to ES for 8 weeks, caused improved healing, as indicated by increased bone formation, strength, vessel density, and osteogenic gene expression. Our results demonstrate that ES significantly increases osteogenic differentiation in vitro and that this effect is translated into improved healing in vivo. These findings support the use of ES to help BTE treatments achieve their full therapeutic potential.

Histological Scoring Method to Assess Bone Healing in Critical Size Bone Defect Models.

Large bone defects are a major problem in trauma and orthopedic surgery. Tissue engineering based treatments have emerged as promising alternatives to traditional bone grafting techniques. Critical size bone defect animal models have been developed and widely used to evaluate and compare therapeutic effectiveness in bone tissue engineering treatments. To measure healing in a given defect after treatment, histological assessment methods are commonly used. These histological methods are typically qualitative and only measure the amount of newly formed bone. In this study, we introduce a new histological scoring method that in addition to new bone formation also measures newly formed "cartilage," "fibrous tissue," and "remnant bone defect size." Using Kappa analysis and interclass correlation analysis, we verified the reliability of our new scoring method. These additional parameters make it possible to differentiate between the hard callus and soft callus phases of healing and, thus, derive more valuable information about the effect different tissue-engineering treatments have on the healing process.

Shrinkage stress and degree of conversion of a dental composite submitted to different photoactivation protocols.

The aim of this study was to evaluate the polymerization stress and degree of conversion of a composite submitted to different photoactivation protocols. The composite Filtek Z350 was placed in the central perforation of a photoelastic disc and polymerized using a LED-based curing unit (BluePhase II--Ivoclar Vivadent) with energy density of 12, 24 or 36 J/cm2 using the following photopolymerization protocols: continuous high intensity (HI: 1200 mW/cm2 during 10, 20 or 30s), continuous low intensity (LI: 650 mW/cmz during 18, 36 or 54s) and soft-start (SS: 150 mW/cm2 during 5 s + 1200 mW/cm2 during 9, 19 or 29s) (n = 5). Photoelastic analysis was used to evaluate polymerization shrinkage stress and FTIR was performed to determine the degree of conversion of the composite. ANOVA 3-way procedure was used to determine the significance of the main effects and their interactions followed by two-way ANOVA for each time was performed (p < 0.05). Shrinkage stress increased with higher values of energy. No statistically significant differences on polymerization shrinkage stress were found between high and low intensity activation modes. Soft-start method generated stresses that were statistically lower than continuous modes except when 12 J/cm2 was applied. Similar degree of conversion was observed for photoactivation modes used, except for soft-start mode with 12, 24 and 36J/cm2 that showed lowest levels of conversion. Energy density and activation mode influenced polymerization shrinkage stress, but no benefit on degree of conversion was observed.

Shrinkage stress and degree of conversion of a dental composite submitted to different photoactivation protocols

The aim of this study was to evaluate the polymerization stress and degree of conversion of a composite submitted to different photoactivation protocols. The composite Filtek Z350 was placed in the central perforation of a photoelastic disc and polymerized using a LED-based curing unit (BluePhase II - IvoclarVivadent) with energy density of 12, 24 or 36 J/cm² using the following photopolymerization protocols: continuous high intensity (HI: 1200 mW/cm² during 10, 20 or 30s), continuous low intensity (LI: 650 mW/cm² during 18, 36 or 54s) and soft-start (SS: 150 mW/cm² during 5 s + 1200 mW/cm² during 9, 19 or 29s) (n=5). Photoelastic analysis was used to evaluate polymerization shrinkage stress and FTIR was performed to determine the degree of conversion of the composite. ANOVA 3-way procedure was used to determine the significance of the main effects and their interactions followed by two-way ANOVA for each time was performed (p<0.05). Shrinkage stress increased with higher values of energy. No statistically significant differences on polymerization shrinkage stress were found between high and low intensity activation modes. Softstart method generated stresses that were statistically lower than continuous modes except when 12 J/cm² was applied. Similar degree of conversion was observed for photoactivation modes used, except for soft-start mode with 12, 24 and 36 J/cm² that showed lowest levels of conversion. Energy density and activation mode influenced polymerization shrinkage stress, but no benefit on degree of conversion was observed.

O objetivo neste estudo foi avaliar a tensão de contração de polimerização e o grau de conversão de uma resina composta submetida a vários protocolos de fotoativação. O compósito Filtek Z350 foi inserido na perfuração central de um disco de resina fotoelástica e polimerizado usando uma unidade de fotoativação LED (BluePhase II - IvoclarVivadent) com as doses de energia de 12, 24 or 36 J/cm² usando os seguintes protocolos de polimerização: contínuo de alta intensidade (HI: 1200 mW/cm² durante 10, 20 ou 30s), contínuo de baixa intensidade (LI: 650 mW/cm² durante 18, 36 ou54s) e "soft-start" (SS: 150 mW/cm² durante 5 s + 1200 mW/cm² durante 9, 19 ou 29s) (n=5). Análise fotoelástica e Espectroscopia Infravermelha Transformada de Fourier (FTIR) foram usados para avaliar a tensão de contração de polimerização e grau de conversão do compósito respectivamente. O teste ANOVA três fatores foi usado para determinar os principais efeitos e interações das variáveis e seguidamente, ANOVA 2 fatores para ver a diferença entre os grupos (p<0.05). A tensão de contração aumentou com valores maiores de energia. Não foram observadas diferenças estatisticamente significantes para a tensão de contração entre os modos de alta e baixa intensidade. O modo "soft-start" gerou menor tensão que os modos contínuos, exceto quando 12 J/cm² foi aplicado. Similar grau de conversão foi observado para os modos de fotoativação usados, com exceção do modo "soft-start" com 12, 24 e 36 J/cm² que mostraram níveis menores de conversão. A dose de energia e modo de ativação influenciam a tensão de contração de polimerização, porém nenhum benefício no grau de conversão foi observado.