Tissue Engineering Part A最新文献

Adequate bone volume is required for osseointegrated implants to restore lost teeth and oral function. Several studies have demonstrated potential advantage of stem cells in regenerative medicine using osteoblasts. The periosteum is composed of osteoblasts, fibroblasts, and osteoprogenitor cells. It may be an alternative source for bone tissue engineering because of easy isolation and rapid proliferation in vivo and in vitro. Low-intensity pulsed ultrasound (LIPUS) has proved successful in recoveries from nonunions, delayed unions, and fracture of the bone in both animal experiments and clinical treatments. The study was to investigate the influence of LIPUS on the osteogenic differentiation in murine periosteum-derived cells (PDCs) and the underlying mechanism of LIPUS. PDCs were treated daily with LIPUS for 20 min up to 21 days with 3 MHz frequency, 30 mW/cm² intensity, and pulse repetition frequency of 1 kHz. The effects of LIPUS on cell proliferation and viability were investigated. Osteogenic differentiation was analyzed by alkaline phosphatase (ALP)-positive cell staining, ALP activity assay, mineralized nodule formation, real-time reverse transcription-polymerase chain reaction, as well as western blotting. The results indicated that ultrasound stimulation did not significantly affect the proliferation of PDCs. But LIPUS significantly increased ALP activity on day 7 and markedly promoted formation of mineralized nodules on day 21. mRNA expression of ALP and osteocalcin was significantly upregulated by stimulation with LIPUS. LIPUS enhanced gene expression of both bone morphogenetic protein-2 (BMP-2) and osterix only in the presence of osteogenic medium. LIPUS stimulation did not affect Smad 1 and Smad 5 protein expression, but significantly upregulated protein levels of BMP-2 and phosphor-Smad 1/5/9 in PDCs. Thus, LIPUS stimulation increased early osteogenic differentiation in a normal medium and further enhanced expression of BMP-2 and subsequent osterix expression through the canonical Smad-signaling pathway in an osteogenic medium, leading to mineral apposition. Therefore, LIPUS might have potential to promote osteogenesis in PDCs. Impact statement There are few studies on periosteum-derived cells (PDCs) because conventional methods of their isolation are relatively difficult to procure abundant cells for cell culture and the total cell numbers are limited. In this study, a modified isolation technique of murine calvarial PDCs using gelatin is described. PDCs were initiated to emerge as early as day 3 and showed increased proliferation, which can be used for further studies. Low-intensity pulsed ultrasound stimulation increased early osteogenic differentiation in a normal medium and further enhanced expression of bone morphogenic protein-2 and subsequent osterix expression through the canonical Smad-signaling pathway in an osteogenic medium, leading to mineral apposition.

Primary closure of fetal skin in spina bifida protects the spinal cord and improves clinical outcome, but is also associated with postnatal growth malformations and spinal cord tethering. In this study, we evaluated the postnatal effects of prenatally closed full-thickness skin defects in sheep applying collagen scaffolds with and without heparin/vascular endothelial growth factor/fibroblast growth factor 2, focusing on skin regeneration and growth. At 6 months, collagen scaffold functionalized with heparin, VEGF, and FGF2 (COL-HEP/GF) resulted in a 6.9-fold increase of the surface area of the regenerated skin opposed to 1.7 × for collagen only. Epidermal thickness increased 5.7-fold at 1 month, in line with high gene expression of S100 proteins, and decreased to 2.1 at 6 months. Increased adipose tissue and reduced scaffold degradation and number of myofibroblasts were observed for COL-HEP/GF. Gene ontology terms related to extracellular matrix (ECM) organization were enriched for both scaffold treatments. In COL-HEP/GF, ECM gene expression resembled native skin. Expression of hair follicle-related genes in COL-HEP/GF was comparable to native skin, and de novo hair follicle generation was indicated. In conclusion, in utero closure of skin defects using functionalized collagen scaffolds resulted in long-term skin regeneration and growth. Functionalized collagen scaffolds that grow with the child may be useful for prenatal treatment of closure defects like spina bifida. Impact statement Prenatal closure of fetal skin in case of spina bifida prevents damage to the spinal cord. Closure of the defect is challenging and may result in postnatal growth malformations. In this study, the postnatal effects of a prenatally applied collagen scaffold functionalized with heparin and vascular endothelial growth factor (VEGF)/fibroblast growth factor (FGF) were investigated. An increase of the surface area of regenerated skin ("growing with the child") and generation of hair follicles was observed. Gene expression levels resembled those of native skin with respect to the extracellular matrix and hair follicles. Overall, in utero closure of skin defects using heparin/VEGF/FGF functionalized collagen scaffolds results in long-term skin regeneration.

To investigate the effect of soluble factors released from human nasal chondrocytes (NCs) on cocultured human bone marrow mesenchymal stem cells (MSCs) and NC tissue-engineered constructs. Cartilage engineered from pure NCs on a three-dimensional (3D) porous collagen scaffold was cultured indirectly in a Transwell system with cartilage engineered from a direct coculture of human bone marrow-derived MSCs and NCs on a 3D porous collagen scaffold. The soluble factors were measured in the conditioned media from the different chambers of the Transwell system. Engineered cartilage from cocultures exposed to the pure NC construct exhibited reduced chondrogenic potential relative to control constructs, shown by reduced extracellular matrix deposition and increased expression of hypertrophic markers. Analysis of the soluble factors within the conditioned media showed an increase in inflammatory cytokines in the coculture chamber exposed to the pure NC construct. Principal component analysis revealed that the majority of the data variance could be explained by proinflammatory factors and hypertrophic chondrogenesis. In conclusion, our data suggest that inflammatory cytokines derived from NCs reduce the chondrogenic potential of coculture engineered cartilage through the induction of hypertrophic chondrogenesis. Impact statement The use of engineered cartilage from cocultured nasal chondrocytes (NCs) and mesenchymal stem cells for nasal cartilage reconstruction may be problematic. Our data suggest that the soluble factors from surrounding native NCs in the cartilage to be fixed can compromise the quality of the engineered cartilage if used in reconstructive surgery.

Erectile dysfunction caused by damage to the cavernous nerve is a common complication of radical prostatectomy for patients with localized prostate cancer. Various studies have investigated repair of damaged tissue and prevention of fibrosis in the corpus cavernosum using stem cell therapy. However, stem cell therapy has limitations, including insufficient nutrient and oxygen supply to transplanted stem cells. This study investigated whether stem cell/oxygen-releasing hollow microparticles (HPs) had therapeutic effect on erectile dysfunction in a rat model of bilateral cavernous nerve injury (BCNI). Therapeutic effects were observed in the BCNI model at 1, 2, and 4 weeks postcavernous nerve injury. Erectile function further improved after treatment with stem cell/oxygen-releasing HP system compared with treatment with only stem cells at 4 weeks. Stem cell/oxygen-releasing HP system increased cyclic guanosine monophosphate (cGMP) level and neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), α-smooth muscle actin (α-SMA), and muscarinic acetylcholine receptor 3 (M3) expression while decreasing fibrosis and apoptosis in the corpus cavernosum. Our results clearly show that stem cell survival increases around transplanted stem cell/oxygen-releasing hybrid system site. Taken together, an oxygen-releasing HP system supported prolonged stem cell survival, sustaining the paracrine effect of the stem cells, and consequently enhancing erectile function. These findings show promise with regard to prolonged stem cell survival in stem cell applications for various diseases and types of tissue damage. Impact statement In this study, we used an oxygen-releasing hollow microparticles (HPs) system with stem cells to attempt to overcome certain limitations of stem cell therapy, including insufficient nutrient and oxygen supplies for transplanted stem cells. Our results demonstrated that a stem cell/oxygen-releasing HP hybrid system could further improve erectile function, cyclic guanosine monophosphate (cGMP) level, and NOS level in a bilateral cavernous nerve injury rat model through prolonged stem cell survival. Our data suggest that a stem cell/oxygen-releasing HP system is a promising clinical treatment option for postprostatectomy erectile dysfunction. Furthermore, this system may be relevant in different disease therapies and regenerative medicine.

With age, adult skeletal muscle (SkM) is known to decrease in muscle mass, strength, and functional capacity, a state known as sarcopenia. Here we developed an in vitro three-dimensional (3D) bioengineered senescent SkM tissue using primary human myoblasts. These tissues exhibited the characteristics of atrophied muscle, including expression of senescent genes, decreased number of satellite cells, reduced number and size of myofibers, and compromised metabolism and calcium flux. As a result, senescent SkM tissues showed impaired ability to generate force in response to electrical stimulation compared with young tissues. Furthermore, in contrast to young SkM tissues, senescent tissues failed to regenerate in response to injury, possibly as a result of persistent apoptosis and failure to initiate a proliferation program. Our findings suggest that 3D senescent SkM may provide a powerful model for studying aging and a platform for drug testing and discovery of therapeutic compounds to improve the function of sarcopenic muscle. Impact statement Skeletal muscle (SkM) plays important physiological roles and has significant regenerative capacity. However, aged SkM lose their functionality and regeneration ability. In this article, we present a senescent human bioengineering SkM tissue model that can be used to investigate senescence, metabolic or genetic diseases that inflict SkM, and to test various strategies including novel small molecules that restore muscle function and promote regeneration. One key limitation of two-dimensional cell culture system is the detachment of contractile myotubes from the surface over time, thereby limiting the evaluation of myogenic function. Here we use primary human myoblasts, which exhibit all major hallmarks of aging to mimic the organization and function of native muscle. Using this system, we were able to measure the contractile function, calcium transients, and regeneration capacity of SkM tissues. We also evaluated the response of senescent SkM tissues to injury and their ability to regenerate and recover, compared with "young" tissues. Our results suggest that three-dimensional constructs enable organization of contractile units including myosin and actin filaments, thereby providing a powerful platform for the quantitative assessment of muscle myotubes in response to injury, genetic or metabolic disorders, or pharmacological testing.

Distraction osteogenesis (DO) is a well-established surgical technique for treating bone defect and limb lengthening. The major drawback of DO is the long treatment period as the external fixator has to be kept in place until consolidation is completed. Calcitonin gene-related peptide (CGRP) has been reported to promote angiogenesis by affecting endothelial progenitor cells (EPCs) in limb ischemia and wound healing. Thus, the goal of this study was to evaluate the angiogenic effect of exogenous CGRP on bone regeneration in a rat DO model. Exogenous CGRP was directly injected into the bone defect after each cycle of distraction in vivo. Microcomputed tomography, biomechanical test, and histological analysis were performed to assess the new bone formation. Angiography and immunofluorescence were performed to assess the formation of blood vessels. CD31⁺CD144⁺ EPCs in the bone defect were quantified with flow cytometry. In in vitro study, bone marrow stem cells (BMSCs) were used to investigate the effect of CGRP on EPCs production during endothelial differentiation. Our results showed that CGRP significantly promoted bone regeneration and vessel formation after consolidation. CGRP significantly increased the fraction of CD31⁺CD144⁺EPCs and the capillary density in the bone defect at the end of distraction phase. CGRP increased EPC population in the endothelial differentiation of BMSCs in vitro by activating PI3K/AKT signaling pathway. Furthermore, differentiated EPCs rapidly assembled into tube-like structures and promoted osteogenic differentiation of BMSCs. In conclusion, CGRP increased EPC population and promoted blood vessel formation and bone regeneration at the defect region in a DO model. Impact statement Distraction osteogenesis (DO) is a well-established surgical technique for limb lengthening and bone defect. The disadvantage of this technique is that external fixator is needed to be kept in place for about 12 months. This may result in increased risk of infection, financial burden, and negative psychological impacts. In this study, we have injected calcitonin gene-related peptide (CGRP) into the defect region after distraction and found that CGRP enhanced vessel formation and bone regeneration in a rat DO model. This suggests that a controlled delivery system for CGRP could be developed and applied clinically for accelerating bone regeneration in patients with DO.

Poly(l-lactide) (PLLA) as one of the most well-known biodegradable polyesters has been studied extensively for bone tissue engineering. If being properly programmed, scaffolds from PLLA can also be endowed with the capability of shape memory. However, several noted issues, for example, mechanical brittleness, high glass transition temperature T_g, and relatively poor shape retention and recovery properties, necessitate modification of the PLLA to improve its application efficacy in physiological conditions. This study is proposed to modify PLLA by having the biodegradable poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) incorporated to form ultrafine composite fibers (i.e., PLLA-PHBV) through electrospinning. Different pairs of PLLA-PHBV at the varying mass ratios of 10:0, 9:1, 8:2, 7:3, 6:4, and 0:10 can be successfully electrospun into fibrous form with the fineness of 2-3 μm. Incorporation of PHBV enables to give rise to desired T_g decreases and also, interestingly, increases in the Young's modulus of the PLLA-PHBV blends, while gradually increasing the PHBV mass ratios up to 30%. The PLLA-PHBV (7:3) formulation is identified to present excellent shape memory properties with high shape fixing ratio (>98%) and shape recovery ratio (>96%) compared to the unmodified PLLA fiber counterpart. Moreover, the PLLA-PHBV (7:3) fibers also show enhanced osteogenesis-inducing ability in the mouse bone mesenchymal stem cells, even under nonosteoinductive conditions. Collectively, for the first time this study demonstrates the enhanced shape memory and osteogenesis capabilities of the electrospun PLLA-PHBV composite fibers, and the researched PLLA-PHBV (7:3) fiber system could be potentially applied as a multifunctional scaffolding material for applications in bone tissue repair and regeneration. Impact statement By first converting the poly(l-lactide) (PLLA)-poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) hybrids into fibrous form at varied mass ratios followed by a thorough characterization, we reasonably demonstrated that incorporation of an appropriate amount of PHBV (i.e., 30%) into the PLLA fibers could give rise to significant improvement on the shape memory capability of the PLLA, along with the desired decreases in the transition temperature (T_g). Moreover, the fibrous PLLA-PHBV (7:3) scaffold was also found to significantly promote the osteogenic commitment in bone mesenchymal stem cells with osteoinductive factors in a synergistic manner. Our biomimicking and shape memory enabled fibrous scaffold of PLLA-PHBV could be used to construct multifunctional three-dimensional scaffold with shape memory effect for bone regeneration.

Various therapeutic approaches, for example, in case of trauma or cancer require the transplantation of autologous tissue. Depending on the size and the origin of the harvested tissue, these therapies can lead to iatrogenic complications and donor-site morbidities. In future, these side effects could be avoided by transplanting artificially generated tissue consisting of different cell types and matrix components derived from the host body. Tissue that is grown in the patient could be advantageous compared with the more simply structured in vitro-grown alternatives. To overcome the limitations of graft vascularization, the arteriovenous (AV) loop technique has been established for different tissues in the last years and was adapted for lymphatic tissue engineering in the present study. We utilized the AV loop technique to grow human lymphatic vasculature in vivo in the Rowett nude (RNU) rat. A combination of human lymphatic endothelial cells (LECs) and bone marrow-derived mesenchymal stem cells was implanted in a fibrin matrix surrounding the AV loop. After 2 or 4 weeks of implantation, the animals were perfused and the tissue was harvested. It could be demonstrated by immunohistochemistry for human LYVE1, human CD31, and murine podoplanin that the implanted cells formed human lymphatic vasculature in the AV loop chamber. Beside development of murine podoplanin-positive vasculature in the AV loop tissue, vasculature positive for human marker proteins developed in comparable numbers. This suggests that implanted LECs are able to improve the lymphatic vascularization of the newly engineered tissue. Thus, we were able to establish an in vivo tissue engineering method to generate lymphatic vascularized soft tissue. An axially vascularized transplantable lymphatic vessel network was engineered without requiring advanced cell culture equipment, rendering the lymphatic AV loop highly suitable for applied regenerative medicine. Impact statement Various surgical procedures require the transplantation of autologous harvested tissue, for example, the vascularized lymph node transfer for the treatment of lymphedema. Tissue-engineered transplants could be used instead of autologous transplants and thereby help to reduce the side effects of those therapies. However, in vitro tissue engineering of large constructs requires a lot of know-how as well as advanced cell culture equipment, which might not be accessible in every hospital. In vivo tissue engineering approaches like the presented technique for the generation of transplantable networks of lymphatic vasculature could serve as an alternative for in vitro tissue engineering approaches in clinical settings.

External volume expansion (EVE) promotes proliferation of adipose-derived stem cells (ADSCs) during adipose tissue regeneration. However, the mechanism by which EVE is translated into biochemical signals and subsequently induces proliferation of ADSCs is poorly understood. Here, we investigated the strain in adipose tissue and mechanochemical signaling upon EVE in rats. In addition, the effect of mechanical strain on proliferation of ADSCs was assessed using a custom-built Flexcell device. The level of strain in adipose tissue upon EVE peaked at week 1 and then decreased over time, and the cell proliferation rate was similarly affected. Mechanical strain-dependent activation of integrin β1 and the RhoA/myosin light chain (MLC) pathway was involved in cell proliferation. The proliferation rate of ADSCs was higher under 12% mechanical strain than under 6% and 0% mechanical strain in vitro. Mechanical strain-dependent activation of integrin β1 promoted activation of the small GTPase RhoA and phosphorylation of MLC. Furthermore, knockdown of integrin β1 attenuated activation of the RhoA/MLC pathway and proliferation of ADSCs in response to mechanical strain. Taken together, this study provides the first evidence of mechanochemical signaling in response to EVE. These data may help elucidate the effects of different strain levels on adipose tissue regeneration. Impact statement External volume expansion (EVE) induces adipose tissue regeneration and has great therapeutic potential to correct soft tissue defects. This study showed that EVE promotes proliferation of adipose-derived stem cells by activating integrin β1 and its crucial downstream signaling molecules, namely the small GTPase RhoA and p-myosin light chain. The findings of this study may assist clinical tissue engineering applications and provide new insights into the regulation of adipose tissue regeneration in clinical practice.

Recent advancements in tissue engineering suggest that biomaterials, such as decellularized extracellular matrix (ECM), could serve to potentiate the localization and efficacy of regenerative therapies in the central nervous system. Still, what factors and which mechanisms are required from these ECM-based biomaterials to exert their effect are not entirely understood. In this study, we use the brain as a novel model to test the effects of particular biochemical and structural properties by evaluating, for the first time, three different sections of the brain (i.e., cortex, cerebellum, and remaining areas) side-by-side and their corresponding decellularized counterparts using mechanical (4-day) and chemical (1-day) decellularization protocols. The three different brain subregions had considerably different initial conditions in terms of cell number and growth factor content, and some of these differences were maintained after decellularization. Decellularized ECM from both protocols was used as a substrate or as soluble factor, in both cases showing good cell attachment and growth capabilities. Interestingly, the 1-day protocol was capable of promoting greater differentiation than the 4-day protocol, probably due to its capacity to remove a similar amount of cell nuclei, while better conserving the biochemical and structural components of the cerebral ECM. Still, some limitations of this study include the need to evaluate the response in other biologically relevant cell types, as well as a more detailed characterization of the components in the decellularized ECM of the different brain subregions. In conclusion, our results show differences in neuronal maturation depending on the region of the brain used to produce the scaffolds. Complex organs such as the brain have subregions with very different initial cellular and biochemical conditions that should be considered for decellularization to minimize exposure to immunogenic components, while retaining bioactive factors conducive to regeneration. [Figure: see text] Impact statement The present study offers new knowledge about the production of decellularized extracellular matrix scaffolds from specific regions of the porcine brain, with a direct comparison of their effect on in vitro neuronal maturation. Our results show differences in neuronal maturation depending on the region of the brain used to produce the scaffolds, suggesting that it is necessary to consider the initial cellular content of the source tissue and its bioactive capacity for the production of an effective regenerative therapy for stroke.