Showing papers in "Tissue Engineering Part A in 2013"
TL;DR: BNFSs provide a versatile scaffold capable of supporting both stem cell expansion and differentiation for T/L tissue engineering applications, and mimicked the normal tri-phasic mechanical behavior of native tendon and ligament (T/L) during loading.
Abstract: Tendon and ligament (T/L) injuries present an important clinical challenge due to their intrinsically poor healing capacity. Natural healing typically leads to the formation of scar-like tissue possessing inferior mechanical properties. Therefore, tissue engineering has gained considerable attention as a promising alternative for T/L repair. In this study, we fabricated braided nanofibrous scaffolds (BNFSs) as a potential construct for T/L tissue engineering. Scaffolds were fabricated by braiding 3, 4, or 5 aligned bundles of electrospun poly(L-lactic acid) nanofibers, thus introducing an additional degree of flexibility to alter the mechanical properties of individual scaffolds. We observed that the Young's modulus, yield stress, and ultimate stress were all increased in the 3-bundle compared to the 4- and 5-bundle BNFSs. Interestingly, acellular BNFSs mimicked the normal tri-phasic mechanical behavior of native tendon and ligament (T/L) during loading. When cultured on the BNFSs, human mesenchymal stem cells (hMSCs) adhered, aligned parallel to the length of the nanofibers, and displayed a concomitant realignment of the actin cytoskeleton. In addition, the BNFSs supported hMSC proliferation and induced an upregulation in the expression of key pluripotency genes. When cultured on BNFSs in the presence of tenogenic growth factors and stimulated with cyclic tensile strain, hMSCs differentiated into the tenogenic lineage, evidenced most notably by the significant upregulation of Scleraxis gene expression. These results demonstrate that BNFSs provide a versatile scaffold capable of supporting both stem cell expansion and differentiation for T/L tissue engineering applications.
170 citations
TL;DR: Examination of scaffolds produced using a variety of detergents indicated that detergent choice influenced human immune response in terms of T cell activation and chemokine production.
Abstract: The authors have previously shown that acellular (AC) trachea-lung scaffolds can (1) be produced from natural rat lungs, (2) retain critical components of the extracellular matrix (ECM) such as collagen-1 and elastin, and (3) be used to produce lung tissue after recellularization with murine embryonic stem cells. The aim of this study was to produce large (porcine or human) AC lung scaffolds to determine the feasibility of producing scaffolds with potential clinical applicability. We report here the first attempt to produce AC pig or human trachea-lung scaffold. Using a combination of freezing and sodium dodecyl sulfate washes, pig trachea-lungs and human trachea-lungs were decellularized. Once decellularization was complete we evaluated the structural integrity of the AC lung scaffolds using bronchoscopy, multiphoton microscopy (MPM), assessment of the ECM utilizing immunocytochemistry and evaluation of mechanics through the use of pulmonary function tests (PFTs). Immunocytochemistry indicated that there was loss of collagen type IV and laminin in the AC lung scaffold, but retention of collagen-1, elastin, and fibronectin in some regions. MPM scoring was also used to examine the AC lung scaffold ECM structure and to evaluate the amount of collagen I in normal and AC lung. MPM was used to examine the physical arrangement of collagen-1 and elastin in the pleura, distal lung, lung borders, and trachea or bronchi. MPM and bronchoscopy of trachea and lung tissues showed that no cells or cell debris remained in the AC scaffolds. PFT measurements of the trachea-lungs showed no relevant differences in peak pressure, dynamic or static compliance, and a nonrestricted flow pattern in AC compared to normal lungs. Although there were changes in content of collagen I and elastin this did not affect the mechanics of lung function as evidenced by normal PFT values. When repopulated with a variety of stem or adult cells including human adult primary alveolar epithelial type II cells both pig and human AC scaffolds supported cell attachment and cell viability. Examination of scaffolds produced using a variety of detergents indicated that detergent choice influenced human immune response in terms of T cell activation and chemokine production.
153 citations
TL;DR: It was observed that scaffold mechanical properties, cell proliferation, matrix production, and differentiation were regulated by changes in the fiber diameter, and it was demonstrated that controlling the scaffold fiber diameter is critical in the design of scaffolds for functional and guided connective tissue repair.
Abstract: The diameter of collagen fibrils in connective tissues, such as tendons and ligaments is known to decrease upon injury or with age, leading to inferior biomechanical properties and poor healing capacity. This study tests the hypotheses that scaffold fiber diameter modulates the response of human tendon fibroblasts, and that diameter-dependent cell responses are analogous to those seen in healthy versus healing tissues. Particularly, the effect of the fiber diameter (320 nm, 680 nm, and 1.80 μm) on scaffold properties and the response of human tendon fibroblasts were determined over 4 weeks of culture. It was observed that scaffold mechanical properties, cell proliferation, matrix production, and differentiation were regulated by changes in the fiber diameter. More specifically, a higher cell number, total collagen, and proteoglycan production were found on the nanofiber scaffolds, while microfibers promoted the expression of phenotypic markers of tendon fibroblasts, such as collagen I, III, V, and tenomodulin. It is possible that the nanofiber scaffolds of this study resemble the matrix in a state of injury, stimulating the cells for matrix deposition as part of the repair process, while microfibers represent the healthy matrix with micron-sized collagen bundles, thereby inducing cells to maintain the fibroblastic phenotype. The results of this study demonstrate that controlling the scaffold fiber diameter is critical in the design of scaffolds for functional and guided connective tissue repair, and provide new insights into the role of matrix parameters in guiding soft tissue healing.
147 citations
TL;DR: Results for the first time demonstrated that MSCs derived from orofacial tissue encapsulated in RGD-modified alginate scaffold show promise for craniofacial bone regeneration.
Abstract: Mesenchymal stem cells (MSCs) provide an advantageous alternative therapeutic option for bone regeneration in comparison to current treatment modalities. However, delivering MSCs to the defect site while maintaining a high MSC survival rate is still a critical challenge in MSC-mediated bone regeneration. Here, we tested the bone regeneration capacity of periodontal ligament stem cells (PDLSCs) and gingival mesenchymal stem cells (GMSCs) encapsulated in a novel RGD- (arginine-glycine-aspartic acid tripeptide) coupled alginate microencapsulation system in vitro and in vivo. Five-millimeter-diameter critical-size calvarial defects were created in immunocompromised mice and PDLSCs and GMSCs encapsulated in RGD-modified alginate microspheres were transplanted into the defect sites. New bone formation was assessed using microcomputed tomography and histological analyses 8 weeks after transplantation. Results confirmed that our microencapsulation system significantly enhanced MSC viability and osteogenic differe...
145 citations
TL;DR: In this article, a study was conducted to determine the bone formation of light-weight titanium-based implants generated by selective laser melting (SLM) for osteoconduction and cell transplantation.
Abstract: The treatment of large bone defects still poses a major challenge in orthopaedic and cranio-maxillofacial surgery One possible solution could be the development of personalized porous titanium-based implants that are designed to meet all mechanical needs with a minimum amount of titanium and maximum osteopromotive properties so that it could be combined with growth factor-loaded hydrogels or cell constructs to realize advanced bone tissue engineering strategies Such implants could prove useful for mandibular reconstruction, spinal fusion, the treatment of extended long bone defects, or to fill in gaps created on autograft harvesting The aim of this study was to determine the mechanical properties and potential of bone formation of light weight implants generated by selective laser melting (SLM) We mainly focused on osteoconduction, as this is a key feature in bone healing and could serve as a back-up for osteoinduction and cell transplantation strategies To that end, defined implants were produced by SLM, and their surfaces were left untreated, sandblasted, or sandblasted/acid etched In vivo bone formation with the different implants was tested throughout calvarial defects in rabbits and compared with untreated defects Analysis by micro computed tomography (μCT) and histomorphometry revealed that all generatively produced porous Ti structures were well osseointegrated into the surrounding bone The histomorphometric analysis revealed that bone formation was significantly increased in all implant-treated groups compared with untreated defects and significantly increased in sand blasted implants compared with untreated ones Bone bridging was significantly increased in sand blasted acid-etched scaffolds Therefore, scaffolds manufactured by SLM should be surface treated Bone augmentation beyond the original bone margins was only seen in implant-treated defects, indicating an osteoconductive potential of the implants that could be utilized clinically for bone augmentation purposes Therefore, designed porous, lightweight structures have potential for bone regeneration and augmentation purposes, especially when complex and patient-specific geometries are essential
142 citations
TL;DR: Findings indicate that TEMR constructs can improve the in vivo functional capacity of the injured musculature at least, in part, by promoting generation of functional skeletal muscle fibers.
Abstract: The frank loss of a large volume of skeletal muscle (i.e., volumetric muscle loss [VML]) can lead to functional debilitation and presents a significant problem to civilian and military medicine. Current clinical treatment for VML involves the use of free muscle flaps and physical rehabilitation; however, neither are effective in promoting regeneration of skeletal muscle to replace the tissue that was lost. Toward this end, skeletal muscle tissue engineering therapies have recently shown great promise in offering an unprecedented treatment option for VML. In the current study, we further extend our recent progress (Machingal et al., 2011, Tissue Eng; Corona et al., 2012, Tissue Eng) in the development of tissue engineered muscle repair (TEMR) constructs (i.e., muscle-derived cells [MDCs] seeded on a bladder acellular matrix (BAM) preconditioned with uniaxial mechanical strain) for the treatment of VML. TEMR constructs were implanted into a VML defect in a tibialis anterior (TA) muscle of Lewis rats and observed up to 12 weeks postinjury. The salient findings of the study were (1) TEMR constructs exhibited a highly variable capacity to restore in vivo function of injured TA muscles, wherein TEMR-positive responders (n=6) promoted an ≈61% improvement, but negative responders (n=7) resulted in no improvement compared to nonrepaired controls, (2) TEMR-positive and -negative responders exhibited differential immune responses that may underlie these variant responses, (3) BAM scaffolds (n=7) without cells promoted an ≈26% functional improvement compared to uninjured muscles, (4) TEMR-positive responders promoted muscle fiber regeneration within the initial defect area, while BAM scaffolds did so only sparingly. These findings indicate that TEMR constructs can improve the in vivo functional capacity of the injured musculature at least, in part, by promoting generation of functional skeletal muscle fibers. In short, the degree of functional recovery observed following TEMR implantation (BAM+MDCs) was 2.3×-fold greater than that observed following implantation of BAM alone. As such, this finding further underscores the potential benefits of including a cellular component in the tissue engineering strategy for VML injury.
133 citations
TL;DR: SHED-CM promoted the migration and differentiation of endogenous NPC, induced vasculogenesis, and ameliorated ischemic brain injury after pMCAO as well as transplantation of DPSC.
Abstract: Regenerative therapy using stem cells is a promising approach for the treatment of stroke. Recently, we reported that dental pulp stem cells (DPSC) ameliorated ischemic tissue injury in the rat brain and accelerated functional recovery after middle cerebral artery occlusion (MCAO). In this study, we investigated the effects of stem cells from human exfoliated deciduous tooth (SHED)-derived conditioned medium (SHED-CM) on permanent MCAO (pMCAO). Adult male Sprague-Dawley rats were subjected to pMCAO. SHED-CM were then administered intranasally, and the motor function and infarct volume were evaluated. Neurogenesis and vasculogenesis were determined using immunochemical markers. The SHED-CM group had more positive signals than the Dulbecco's modified Eagle's medium group, with doublecortin (DCX), neurofilament H, neuronal nuclei, and rat endothelial cell antigen observed in the peri-infarct area. Migration of neuronal progenitor cells (NPC) with DCX from the subventricular zone to the peri-infarct area was observed on days 6 and 16, with migration on day 6 being the most prominent. In conclusion, SHED-CM promoted the migration and differentiation of endogenous NPC, induced vasculogenesis, and ameliorated ischemic brain injury after pMCAO as well as transplantation of DPSC.
128 citations
TL;DR: The results suggest that human placenta-derived ECM sheets provide a microenvironment favorable to the growth and differentiation of cells, and positive modulate the healing of full-thickness wounds.
Abstract: The human placenta, a complex organ, which facilitates exchange between the fetus and the mother, contains abundant extracellular matrix (ECM) components and well-preserved endogenous growth factors. In this study, we designed a new dermal substitute from human placentas for full-thickness wound healing. Highly porous, decellularized ECM sheets were fabricated from human placentas via homogenization, centrifugation, chemical and enzymatic treatments, molding, and freeze-drying. The physical structure and biological composition of human placenta-derived ECM sheets dramatically supported the regeneration of full-thickness wound in vivo. At the early stage, the ECM sheet efficiently absorbed wound exudates and tightly attached to the wound surface. Four weeks after implantation, the wound was completely closed, epidermic cells were well arranged and the bilayer structure of the epidermis and dermis was restored. Moreover, hair follicles and microvessels were newly formed in the ECM sheet-implanted wounds. Overall, the ECM sheet produced a dermal substitute with similar cellular organization to that of normal skin. These results suggest that human placenta-derived ECM sheets provide a microenvironment favorable to the growth and differentiation of cells, and positive modulate the healing of full-thickness wounds.
125 citations
TL;DR: It is found that CD90(+) cells are more capable of forming bone both in vitro and in vivo, and may be a more effective marker than CD105 to isolate a highly osteogenic subpopulation for bone tissue engineering.
Abstract: Background: Stem cell-based bone tissue engineering with adipose-derived stromal cells (ASCs) has shown great promise for revolutionizing treatment of large bone deficits. However, there is still a lack of consensus on cell surface markers identifying osteoprogenitors. Fluorescence-activated cell sorting has identified a subpopulation of CD105low cells with enhanced osteogenic differentiation. The purpose of the present study was to compare the ability of CD90 (Thy-1) to identify osteoprogenitors relative to CD105. Methods: Unsorted cells, CD90+, CD90−, CD105high, and CD105low cells were treated with an osteogenic differentiation medium. For evaluation of in vitro osteogenesis, alkaline phosphatase (ALP) staining and alizarin red staining were performed at 7 days and 14 days, respectively. RNA was harvested after 7 and 14 days of differentiation, and osteogenic gene expression was examined by quantitative real-time polymerase chain reaction. For evaluation of in vivo osteogenesis, critical-sized (4-mm) ca...
119 citations
TL;DR: It is demonstrated that mild HS treatment may be potentially used to enhance the bone regeneration using hMSCs and enhanced the maturation of osteoblasts differentiated from hMSC.
Abstract: The mechanisms for the heat-induced osteogenesis are not completely known and the thermal regulation of human mesenchymal stem cell (hMSC) differentiation is not well studied. In this study, the di...
108 citations
TL;DR: The TE approach used in this study significantly reduced the number of inflammatory cells around the implanted mesh and promoted neovascularization, which improves mesh biocompatibility and may be an alternative option for future treatment of POP.
Abstract: Background: Pelvic organ prolapse (POP) is defined as the descent of one or more of the pelvic structures into the vagina and includes uterine, vaginal vault, and anterior or posterior vaginal wall prolapse. The treatment of POP may include implantation of a synthetic mesh. However, the long-term benefit of mesh surgery is controversial due to complications such as mesh exposure or pain. The aim of this study was to use a tissue engineering (TE) approach to assess the in vivo biological and biomechanical behavior of a new gelatin/polyamide mesh, seeded with a novel source of mesenchymal stem cells in a subcutaneous rat model of wound repair.
Methods: W5C5-enriched human endometrial mesenchymal stem cells (eMSC) were seeded onto meshes (gelatin-coated polyamide knit) at 100,000 cells/cm2. Meshes, with or without cells were subcutaneously implanted dorsally in immunocompromised rats for 7, 30, 60, and 90 days. Flow cytometry was used to detect DiO labeled cells after explantation. Immunohistochemical assessment of foreign body reaction and tissue integration were conducted. Total collagen and the levels of collagens type III and type I were determined. Uniaxial tensiometry was performed on explanted meshes, originally seeded with and without cells, at days 7 and 90.
Results: Implanted meshes were well tolerated, with labeled cells detected on the mesh up to 14 days postimplantation. Meshes with cells promoted significantly more neovascularization at 7 days (p<0.05) and attracted fewer macrophages at 90 days (p<0.05). Similarly, leukocyte infiltration was significantly lower in the cell-seeded meshes at 90 days (p<0.05). Meshes with cells were generally less stiff than those without cells, after 7 and 90 days implantation.
Conclusion: The TE approach used in this study significantly reduced the number of inflammatory cells around the implanted mesh and promoted neovascularization. Seeding with eMSC exerts an anti-inflammatory effect and promotes wound repair with new tissue growth and minimal fibrosis, and produces mesh with greater extensibility. Cell seeding onto polyamide/gelatin mesh improves mesh biocompatibility and may be an alternative option for future treatment of POP.
TL;DR: Results show that engineered leaflets, while having similar tensile properties and collagen content compared to native leaflets, have better recellularization potential.
Abstract: Tissue-engineered heart valves (TEHV) have been proposed as a promising solution for the clinical needs of pediatric patients. In vivo studies have shown TEHV leaflet contraction and regurgitation after several months of implantation. This has been attributed to contractile cells utilized to produce the extracellular matrix (ECM) during TEHV culture. Here, we utilized such cells to develop a mature ECM in a fibrin-based scaffold that generates commissural alignment in TEHV leaflets and then removed these cells using detergents. Further, we evaluated recellularization with potentially noncontractile cells. A tissue-engineered leaflet model was developed with mechanical anisotropy and tensile properties comparable to an ovine pulmonary valve leaflet. No change in tensile properties occurred after decellularization using 1% sodium dodecyl sulfate and 1% Triton detergent treatment. Cell removal was verified by DNA quantitation and western blot analysis for cellular proteins. Histological and scanning electron microscope imaging showed no significant change in the ECM organization and microstructure. We further tested the recellularization potential of decellularized leaflets by seeding human mesenchymal stem cells (hMSC) on the surface of the leaflets and evaluated them at 1 and 3 weeks in two culture conditions. One medium (M1) was chosen to maintain the MSC phenotype while a second medium (M2) was used to potentially differentiate cells to an interstitial cell phenotype. Cellular quantitation showed that the engineered leaflets were recellularized to the highest concentration with M2 followed by M1, with minimum cell invasion of decellularized native leaflets. Histology showed cellular invasion throughout the thickness of the leaflets in M2 and partial invasion in M1. hMSC stained positive for MSC markers, but also for α-smooth muscle actin in both media at 1 week, with no presence of MSC markers at 3 weeks with the exception of CD90. These results show that engineered leaflets, while having similar tensile properties and collagen content compared to native leaflets, have better recellularization potential.
TL;DR: It is demonstrated that ibuprofen-loaded PELA fibrous membranes prevent peritendinous adhesion formation better than membranes that do not contain ib uprofen, through anti-adhesion and anti-inflammatory actions.
Abstract: Physical barriers are commonly used to reduce peritendinous adhesion after injury. However, the inflammatory response to surgery cannot be prevented. This study was designed to evaluate the ability of ibuprofen-loaded poly(l-lactic acid)-polyethylene glycol (PELA) diblock copolymer fibrous membranes in preventing adhesion formation and reduce inflammation. Electrospun PELA fibrous membranes underwent mechanical testing and were characterized by morphology, surface wettability, drug release, and degradation. Results of an in vitro drug release study showed that a burst release was followed by sustained release from fibrous membranes with high initial ibuprofen content. Fewer L929 mouse fibroblasts adhered to and proliferated on the ibuprofen-loaded PELA fibrous membrane compared with tissue culture plates or PELA fibrous membrane without ibuprofen. In a chicken model of flexor digitorum profundus tendon surgery, the ibuprofen-loaded PELA fibrous membranes prevented tissue adhesion and significantly reduced inflammation. Taken together, these results demonstrate that ibuprofen-loaded PELA fibrous membranes prevent peritendinous adhesion formation better than membranes that do not contain ibuprofen, through anti-adhesion and anti-inflammatory actions.
TL;DR: Findings support the potential use of a moderately crosslinked CDM as a contraction-free biomaterial for cartilage tissue engineering and investigate the ability of chemical crosslinking using a natural biologic crosslinker, genipin, to prevent scaffold contraction while preserving the chondrogenic potential of CDM.
Abstract: Autologous cell-based tissue engineering using three-dimensional scaffolds holds much promise for the repair of cartilage defects. Previously, we reported on the development of a porous scaffold derived solely from native articular cartilage, which can induce human adipose-derived stem cells (ASCs) to differentiate into a chondrogenic phenotype without exogenous growth factors. However, this ASC-seeded cartilage-derived matrix (CDM) contracts over time in culture, which may limit certain clinical applications. The present study aimed to investigate the ability of chemical crosslinking using a natural biologic crosslinker, genipin, to prevent scaffold contraction while preserving the chondrogenic potential of CDM. CDM scaffolds were crosslinked in various genipin concentrations, seeded with ASCs, and then cultured for 4 weeks to evaluate the influence of chemical crosslinking on scaffold contraction and ASC chondrogenesis. At the highest crosslinking degree of 89%, most cells failed to attach to the scaffolds and resulted in poor formation of a new extracellular matrix. Scaffolds with a low crosslinking density of 4% experienced cell-mediated contraction similar to our original report on noncrosslinked CDM. Using a 0.05% genipin solution, a crosslinking degree of 50% was achieved, and the ASC-seeded constructs exhibited no significant contraction during the culture period. Moreover, expression of cartilage-specific genes, synthesis, and accumulation of cartilage-related macromolecules and the development of mechanical properties were comparable to the original CDM. These findings support the potential use of a moderately (i.e., approximately one-half of the available lysine or hydroxylysine residues being crosslinked) crosslinked CDM as a contraction-free biomaterial for cartilage tissue engineering.
TL;DR: The approach of combining substrate cues with growth factor augmentation offers a new means to engineer well-organized, collagen-based constructs with an appropriate nanoscale structure for corneal repair and regeneration.
Abstract: Recapitulating the microstructure of the native human corneal stromal tissue is believed to be a key feature in successfully engineering the corneal tissue. The stratified multilayered collagen fibril lamellae with orthogonal orientation determine the robust biomechanical properties of this tissue, and the uniform collagen fibril size and interfibrillar spacing are critical to its optical transparency. The objective of this investigation was to develop a highly organized collagen-fibril construct secreted by human corneal stromal stem cells (hCSSCs) to mimic the human corneal stromal tissue. In culture on a highly aligned fibrous substrate made from poly(ester urethane) urea, the fibroblast growth factor-2 (FGF-2, 10 ng/mL) and transforming growth factor-beta 3 (TGF-β3, 0.1 ng/mL) impacted the organization and abundance of the secreted collagen fibril matrix. hCSSCs differentiated into keratocytes with significant upregulation of the typical gene markers, including KERA, B3GnT7, and CHST6. FGF-2 treatment...
TL;DR: The use of tendon-derived extracellular matrix (TDM)-coated electrospun multilayered scaffolds compared to fibronectin (FN) or phosphate-buffered saline (PBS) coating for use in rotator cuff tendon tissue engineering is investigated to suggest that nonaligned multi-layered electrospuns permit tenogenic differentiation by hASCs and that TDM may promote some aspects of this differentiation.
Abstract: Full-thickness rotator cuff tears are one of the most common causes of shoulder pain in people over the age of 65. High retear rates and poor functional outcomes are common after surgical repair, and currently available extracellular matrix scaffold patches have limited abilities to enhance new tendon formation. In this regard, tissue-engineered scaffolds may provide a means to improve repair of rotator cuff tears. Electrospinning provides a versatile method for creating nanofibrous scaffolds with controlled architectures, but several challenges remain in its application to tissue engineering, such as cell infiltration through the full thickness of the scaffold as well as control of cell growth and differentiation. Previous studies have shown that ligament-derived extracellular matrix may enhance differentiation toward a tendon or ligament phenotype by human adipose stem cells (hASCs). In this study, we investigated the use of tendon-derived extracellular matrix (TDM)-coated electrospun multilayered scaff...
TL;DR: In this special edition, an attempt is made to elucidate the advantages of plant-derived human recombinant collagen and its applications in tissue engineering, particularly skin and wound healing.
Abstract: Extracellular matrix (ECM)-based scaffolds, through their inherent bioactivity and molecular recognition signals, provide the ideal substrate for tissue engineering and regenerative applications. Collagen, the most abundant ECM protein, has proven itself to be a very versatile material with applications in many fields, including the leather and food industries, cosmetics, drug delivery, and tissue engineering. However, doubts persist about the optimal source of collagen for tissue engineering applications, given possible immunogenicity and disease transmission associated with animal sources and reduced bioactivity and availability of recombinant technologies. In this special edition, an attempt is made to elucidate the advantages of plant-derived human recombinant collagen and its applications in tissue engineering, particularly skin and wound healing. While results are promising, the widespread use of animal-derived collagen means that recombinant technologies may find applications in niche areas.
TL;DR: It is concluded that human ASCs promote tissue growth and angiogenesis in the rat vascularized chamber, thereby showing promise for tissue-engineering applications for regenerative therapy.
Abstract: Adult mesenchymal stem cells secrete a variety of angiogenic cytokines and growth factors, so we proposed that these paracrine mechanisms may be used to promote vascularization and growth for tissue engineering in vivo We tested whether or not human adipose-derived stem cells (ASCs) promote tissue formation in rats ASCs were evaluated in vitro for mRNA expression of angiogenic factors, including the vascular endothelial growth factor, basic fibroblast growth factor, interleukin-8 (IL-8), and stromal cell-derived factor-1 (SDF-1) and proliferative activity on human microvascular endothelial cells For in vivo analysis, CM-DiI-labeled ASCs were implanted with a rat cardiac extracellular matrix (ECM) extract-derived hydrogel into a chamber with a femoral arteriovenous loop in the groin of male nude rats for 7 days Vascularization in newly generated tissue was estimated by histomorphometry after endothelial nitric oxide synthase (eNOS) immunostaining ASCs expressed growth factor mRNA and produced an angiogenic activity in vitro After implantation, ASCs survived, but remained suspended in the ECM and relatively few were incorporated into the newly formed tissue The volume of newly generated tissue was significantly higher in chambers containing ASCs and it was enriched with vasculature when compared with the ECM alone We conclude that human ASCs promote tissue growth and angiogenesis in the rat vascularized chamber, thereby showing promise for tissue-engineering applications for regenerative therapy
TL;DR: In conclusion, incorporation of nanostructured colloidal gelatin gels capable of time- and dose-controlled delivery of BMP-2 and FGF-2 in porous titanium scaffolds is a promising strategy to enhance and continue bone regeneration of large bone defects.
Abstract: Porous titanium scaffolds are a promising class of biomaterials for grafting large bone defects, because titanium provides sufficient mechanical support, whereas its porous structure allows bone ingrowth resulting in good osseointegration. To reinforce porous titanium scaffolds with biological cues that enhance and continue bone regeneration, scaffolds can be incorporated with bioactive gels for time- and dose-controlled delivery of multiple growth factors (GFs). In this study, critical femoral bone defects in rats were grafted with porous titanium scaffolds incorporated with nanostructured colloidal gelatin gels. Gels were loaded with bone morphogenetic protein-2 (BMP-2, 3 mg), fibroblast growth factor-2 (FGF-2, 0.6 mg), BMP-2, and FGF-2 (BMP-2/FGF-2, ratio 5:1) or were left unloaded. GF delivery was controlled by fine tuning the crosslinking density of oppositely charged nanospheres. Grafted femurs were evaluated using in vivo and ex vivo micro-CT, histology, and three-point bending tests. All porous titanium scaffolds containing GF-loaded gels accelerated and enhanced bone regeneration: BMP-2 gels gave an early increase (0–4 weeks), and FGF-2 gels gave a late increase (8–12 weeks). Interestingly, stimulatory effects of 0.6 mg FGF-2 were similar to a fivefold higher dose of BMP-2 (3 mg). BMP-2/FGF-2 gels gave more bone outside the porous titanium scaffolds than gels with only BMP-2 or FGF-2, resulted in bridging of most defects and showed superior bone-implant integrity in three-point bending tests. In conclusion, incorporation of nanostructured colloidal gelatin gels capable of time- and dose-controlled delivery of BMP-2 and FGF-2 in porous titanium scaffolds is a promising strategy to enhance and continue bone regeneration of large bone defects.
TL;DR: The findings support the concept that the platelet products have a direct beneficial effect on articular chondrocytes and could drive in sequence a transient activation and the resolution of the inflammatory process, thus providing a rational for their use as therapeutic agents in cartilage inflammation and damage.
Abstract: Platelet-rich plasma (PRP), a cocktail of platelet growth factors and bioactive proteins, has been proposed as a therapeutic agent to restore damaged articular cartilage. We report the biological effect of the platelet lysate (PL), a PRP derivative, on primary human articular chondrocytes cultured under both physiological and inflammatory conditions. When added to the culture medium, PL induced a strong mitogenic response in the chondrocytes. The in vitro expanded cell population maintained a chondrogenic redifferentiation potential as revealed by micromass culture in vitro and ectopic cartilage formation in vivo. Further, in chondrocytes cultured in the presence of the proinflammatory cytokine interleukin-1α (IL-1α), the PL induced a drastic enhancement of the synthesis of the cytokines IL-6 and IL-8 and of neutrophil-gelatinase associated lipocalin, a lipocalin expressed during chondrocyte differentiation and inflammation. These events were mediated by the p38 MAP kinase and NF-κB pathways. We observed ...
TL;DR: PP is an effective material for the preservation of sockets with buccal dehiscence and under more severe conditions for bone formation, the growth factors released from platelets had a negative effect on bone formation.
Abstract: Alveolar bone resorption generally occurs during healing after tooth extraction. This study aimed to evaluate the effects of platelet-poor plasma (PPP), platelet-rich plasma (PRP), and platelet-rich fibrin (PRF) on healing in a ridge-augmentation model of the canine socket with dehiscence of the buccal wall. The third mandibular premolars of 12 beagle dogs were extracted and a 3 mm buccal dehiscence from the alveolar crest to the buccal wall of the extraction socket was created. These sockets were then divided into four groups on the basis of the material used to fill the sockets: PPP, PRP, PRF, and control (no graft material) groups. Results were evaluated at 4 and 8 weeks after surgery. The ultrastructural morphology and constructs of each blood product were studied by a scanning electron microscope (SEM) or calculating concentrations of platelets, fibrinogen, platelet-derived growth factor, and transforming growth factor-β. A total of five microcomputed tomography images of specimens were selected for measurement, and the area occupied by the newly formed bone as well as the horizontal bone width were measured. Moreover, decalcified tissue specimens from each defect were analyzed histologically. The median area of new bone at 4 and 8 weeks and median horizontal bone width at 8 weeks were the highest in the PPP group. However, bone maturation in the PRF and the PRP groups was more progressed than that in the PPP and control groups. By SEM findings, the PRF group showed a more highly condensed fibrin fiber network that was regularly arranged when compared with the PPP and PRP groups. The growth factors released from platelets in PRP indicated higher concentrations than that in PRF. Under more severe conditions for bone formation, as in this experiment, the growth factors released from platelets had a negative effect on bone formation. This study showed that PPP is an effective material for the preservation of sockets with buccal dehiscence.
TL;DR: The results of this study indicate that surface treatment of PLLA microfibrous scaffolds with mild Ar or Ar-NH3/H2 plasmas may have important implications in tissue engineering.
Abstract: Poly(l-lactide) (PLLA) microfibrous scaffolds produced by electrospinning were treated with mild Ar or Ar-NH3/H2 plasmas to enhance cell attachment, growth, and infiltration. Goniometry, atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS) measurements were used to evaluate the modification of the scaffold surface chemistry by plasma treatment. AFM and XPS measurements showed that both plasma treatments increased the hydrophilicity without affecting the integrity of the fibrous structure and the fiber roughness, whereas Ar-NH3/H2 plasma treatment also resulted in surface functionalization with amine groups. Culture studies of bovine aorta endothelial cells and bovine smooth muscle cells on the plasma-treated PLLA scaffolds revealed that both Ar and Ar-NH3/H2 plasma treatments promoted cell spreading during the initial stage of cell attachment and, more importantly, increased the cell growth rate, especially for Ar plasma treatment. In vitro cell infiltration studies showed that both plasma treatments effectively enhanced cell migration into the microfibrous scaffolds. In vivo experiments involving the subcutaneous implantation of plasma-treated PLLA scaffolds under the skin of Sprague-Dawley rats also showed increased cell infiltration. The results of this study indicate that surface treatment of PLLA microfibrous scaffolds with mild Ar or Ar-NH3/H2 plasmas may have important implications in tissue engineering. Further modifications with bioactive factors should improve the functions of the scaffolds for specific applications.
TL;DR: Histological and mechanical examinations revealed that iPSC-NCSCs treatment significantly enhanced tendon healing as indicated by the improvement in matrix synthesis and mechanical properties.
Abstract: Induced pluripotent stem cells (iPSCs) hold great potential for cell therapy and tissue engineering. Neural crest stem cells (NCSCs) are multipotent that are capable of differentiating into mesenchymal lineages. In this study, we investigated whether iPSC-derived NCSCs (iPSC-NCSCs) have potential for tendon repair. Human iPSC-NCSCs were suspended in fibrin gel and transplanted into a rat patellar tendon window defect. At 4 weeks post-transplantation, macroscopical observation showed that the repair of iPSC-NCSC-treated tendons was superior to that of non-iPSC-NCSC-treated tendons. Histological and mechanical examinations revealed that iPSC-NCSCs treatment significantly enhanced tendon healing as indicated by the improvement in matrix synthesis and mechanical properties. Furthermore, transplanted iPSC-NCSCs produced fetal tendon-related matrix proteins, stem cell recruitment factors, and tenogenic differentiation factors, and accelerated the host endogenous repair process. This study demonstrates a potential strategy of employing iPSC-derived NCSCs for tendon tissue engineering.
TL;DR: The findings demonstrated that different progenitor populations exist within discrete niches of the Achilles tendon-tendon proper versus peritenon, and supported the hypothesis that the progenitors pools from both regions have distinct properties and contain enriched progensitor subpopulations of different origins.
Abstract: Specific niches may affect how cells from different regions contribute to tendon biology, particularly in regard to the healing of certain tendinopathies. The objectives of this study are to determ...
TL;DR: This study highlights the potential of novel PPy-coated PLA scaffolds in bone tissue engineering by using chondroitin sulfate (CS)-doped PPy coating to modify initially electrically insulating polylactide resulting in novel osteogenic scaffolds.
Abstract: An electrically conductive polypyrrole (PPy) doped with a bioactive agent is an emerging functional biomaterial for tissue engineering. We therefore used chondroitin sulfate (CS)-doped PPy coating to modify initially electrically insulating polylactide resulting in novel osteogenic scaffolds. In situ chemical oxidative polymerization was used to obtain electrically conductive PPy coating on poly-96L/4D-lactide (PLA) nonwoven scaffolds. The coated scaffolds were characterized and their electrical conductivity was evaluated in hydrolysis. The ability of the coated and conductive scaffolds to enhance proliferation and osteogenic differentiation of human adipose stem cells (hASCs) under electrical stimulation (ES) in three-dimensional (3D) geometry was compared to the noncoated PLA scaffolds. Electrical conductivity of PPy-coated PLA scaffolds (PLA-PPy) was evident at the beginning of hydrolysis, but decreased during the first week of incubation due to de-doping. PLA-PPy scaffolds enhanced hASC proliferation significantly compared to the plain PLA scaffolds at 7 and 14 days. Furthermore, the alkaline phosphatase (ALP) activity of the hASCs was generally higher in PLA-PPy seeded scaffolds, but due to patient variation, no statistical significance could be determined. ES did not have a significant effect on hASCs. This study highlights the potential of novel PPy-coated PLA scaffolds in bone tissue engineering.
TL;DR: An intramuscular ectopic bone model is used to develop the translational potential of the original findings using PSCs (as a combination of pericytes and adventitial cells) from human white adipose tissue and evaluated human PSC-mediated bone formation and vascularization in vivo.
Abstract: An ideal mesenchymal stem cell (MSC) source for bone tissue engineering has yet to be identified. Such an MSC population would be easily harvested in abundance, with minimal morbidity and with high purity. Our laboratories have identified perivascular stem cells (PSCs) as a candidate cell source. PSCs are readily isolatable through fluorescent-activated cell sorting from adipose tissue and have been previously shown to be indistinguishable from MSCs in the phenotype and differentiation potential. PSCs consist of two distinct cell populations: (1) pericytes (CD146+, CD34−, and CD45−), which surround capillaries and microvessels, and (2) adventitial cells (CD146−, CD34+, and CD45−), found within the tunica adventitia of large arteries and veins. We previously demonstrated the osteogenic potential of pericytes by examining pericytes derived from the human fetal pancreas, and illustrated their in vivo trophic and angiogenic effects. In the present study, we used an intramuscular ectopic bone model to develop ...
TL;DR: In this paper, a miniaturized fibrin-based EHT format was expanded to a larger six-well format with six flexible silicone posts holding each EHT (15×25×3 mm3).
Abstract: In engineered heart tissues (EHT), oxygen and nutrient supply via mere diffusion is a likely factor limiting the thickness of cardiac muscle strands. Here, we report on a novel method to in vitro perfuse EHT through tubular channels. Adapting our previously published protocols, we expanded a miniaturized fibrin-based EHT-format to a larger six-well format with six flexible silicone posts holding each EHT (15×25×3 mm3). Thin dry alginate fibers (17×0.04×0.04 mm) were embedded into the cell–fibrin–thrombin mix and, after fibrin polymerization, dissolved by incubation in alginate lyase or sodium citrate. Oxygen concentrations were measured with a microsensor in 14-day-old EHTs (37°C, 21% oxygen) and ranged between 9% at the edges and 2% in the center of the tissue. Perfusion rapidly increased it to 10%–12% in the immediate vicinity of the microchannel. Continuous perfusion (20 μL/h, for 3 weeks) of the tubular lumina (100–500 μm) via hollow posts of the silicone rack increased mean dystrophin-positive cardio...
TL;DR: Immunofluorescent staining for hepatic markers, albumin and E-cadherin, confirmed that softer gels promoted better maintenance of the hepatic phenotype, pointing to the importance of substrate mechanical properties on hepatocyte function.
Abstract: In the liver, hepatocytes are exposed to a large array of stimuli that shape hepatic phenotype. This in vivo microenvironment is lost when hepatocytes are cultured in standard cell cultureware, making it challenging to maintain hepatocyte function in vitro. Our article focused on one of the least studied inducers of the hepatic phenotype-the mechanical properties of the underlying substrate. Gel layers comprised of thiolated heparin (Hep-SH) and diacrylated poly(ethylene glycol) (PEG-DA) were formed on glass substrates via a radical mediated thiol-ene coupling reaction. The substrate stiffness varied from 10 to 110 kPa by changing the concentration of the precursor solution. ELISA analysis revealed that after 5 days, hepatocytes cultured on a softer heparin gel were synthesizing five times higher levels of albumin compared to those on a stiffer heparin gel. Immunofluorescent staining for hepatic markers, albumin and E-cadherin, confirmed that softer gels promoted better maintenance of the hepatic phenotype. Our findings point to the importance of substrate mechanical properties on hepatocyte function.
TL;DR: This study showed the safety and efficacy of CGS impregnated with bFGF in the treatment of chronic skin ulcers and showed this combination therapy could be a promising therapy for chronic skin Ulcers.
Abstract: Chronic skin ulcers such as diabetic ulcers and venous leg ulcers are increasing and are a costly problem in healthcare. We have developed a novel artificial dermis, collagen/gelatin sponge (CGS), which is capable of sustained release of basic fibroblast growth factor (bFGF) for more than 10 days. The objective of this study was to investigate the safety and efficacy of CGS impregnated with bFGF in the treatment of chronic skin ulcers. Patients with chronic skin ulcers that had not healed in at least 4 weeks were treated with CGS impregnated with bFGF at 7 or 14 μg/cm(2) after debridement, and the wound bed improvement was assessed 14 days after application. Wound bed improvement was defined as a granulated and epithelialized area on day 14 with a proportion to the baseline wound area after debridement of 50% or higher. The wound area, the wound area on day 14, and the granulation area on day 14 were independently measured by blinded reviewers in a central review using digital images of wounds taken with a calibrator. Patients were followed up until 28 days after application to observe the adverse reactions related to the application of CGS. From May 2010 to June 2011, 17 patients were enrolled and, in 16 patients, the wound bed improved. Among the randomized patients in step 2, no significant difference was seen between the low-dose group and the high-dose group. No serious adverse reactions were observed. Adverse reactions with a clear causal relationship to the study treatment were mild and patients quickly recovered from them. This study is the first-in-man clinical trial of CGS and showed the safety and efficacy of CGS impregnated with bFGF in the treatment of chronic skin ulcers. This combination therapy could be a promising therapy for chronic skin ulcers.
TL;DR: Negatively charged inorganic hydroxyapatite nanoparticles were assembled with positively charged organic poly(d,l-lactic-co-glycolic acid) (PLGA) NPs to create a cohesive colloidal gel, which showed a well-organized, three-dimensional porous structure and could recover after being sheared.
Abstract: Injectable bone fillers have emerged as an alternative to the invasive surgery often required to treat bone defects. Current bone fillers may benefit from improvements in dynamic properties such as shear thinning during injection and recovery of material stiffness after placement. Negatively charged inorganic hydroxyapatite (HAp) nanoparticles (NPs) were assembled with positively charged organic poly(d,l-lactic-co-glycolic acid) (PLGA) NPs to create a cohesive colloidal gel. This material is held together by electrostatic forces that may be disrupted by shear to facilitate extrusion, molding, or injection. Scanning electron micrographs of the dried colloidal gels showed a well-organized, three-dimensional porous structure. Rheology tests revealed that certain colloidal gels could recover after being sheared. Human umbilical cord mesenchymal stem cells were also highly viable when seeded on the colloidal gels. HAp/PLGA NP colloidal gels offer an attractive scheme for injectable filling and regeneration of bone tissue.