Porcine Bladder Acellular Matrix Research Articles

Optimized Biomanufacturing for Treatment of Volumetric Muscle Loss Enables Physiomimetic Recovery.

Volumetric muscle loss (VML) injuries are defined by loss of sufficient skeletal muscle to produce persistent deficits in muscle form and function, with devastating lifelong consequences to both soldiers and civilians. There are currently no satisfactory treatments for VML injuries. The work described herein details the implementation of a fully enclosed bioreactor environment (FEBE) system that efficiently interfaces with our existing automated bioprinting and advanced biomanufacturing methods for cell deposition on sheet-based scaffolds for our previously described tissue-engineered muscle repair (TEMR) technology platform. Briefly, the TEMR technology consists of a porcine bladder acellular matrix seeded with skeletal muscle progenitor cells and preconditioned via 10% uniaxial cyclic stretch in a bioreactor. Overall, TEMR implantation in an established rat tibialis anterior (TA) VML injury model can result in 60 to ∼90% functional recovery. However, our original study documented >50% failure rate. That is, more than half of the implanted TEMR constructs produced no functional improvement beyond no treatment/repair. The high failure rate was attributed to the untoward mechanical disruption of TEMR during surgical implantation. In a follow-up study, adjustments were made to the geometry of both the VML injury and the TEMR construct, and the "nonresponder" group was reduced from over half the TEMR-treated animals to just 33%. Nonetheless, additional improvement is needed for clinical applicability. The main objectives of the current study were twofold: (1) explore the use of advanced biomanufacturing methods (i.e., FEBE bioreactor) to further improve TEMR reliability (i.e., increase functional response rate), (2) determine if previously established bioprinting methods, when coupled to the customized FEBE system would further improve the rate, magnitude or amplitude of functional outcomes following TEMR implantation in the same rat TA VML injury model. The current study demonstrates the unequivocal benefits of a customized bioreactor system that reduces manipulation of TEMR during cell seeding and maturation via bioprinting while simultaneously maximizing TEMR stability throughout the biofabrication process. This new biomanufacturing strategy not only accelerated the rate of functional recovery, but also eliminated all TEMR failures. In addition, implementation of bioprinting resulted in more physiomimetic skeletal muscle characteristics of repaired muscle tissue.

Autologous Endothelial Progenitor Cells and Bioactive Factors Improve Bladder Regeneration.

Insufficient vascularization is still a challenge that impedes bladder tissue engineering and results in unsatisfied smooth muscle regeneration. Since bladder regeneration is a complex articulated process, the aim of this study is to investigate whether combining multiple pathways by exploiting a combination of biomaterials, cells, and bioactive factors, contributes to the improvements of smooth muscle regeneration and vascularization in tissue-engineered bladder. Autologous endothelial progenitor cells (EPCs) and bladder smooth muscle cells (BSMCs) are cultured and incorporated into our previously prepared porcine bladder acellular matrix (BAM) for bladder augmentation in rabbits. Simultaneously, exogenous vascular endothelial growth factor (VEGF) and platelet-derived growth factor BB (PDGF-BB) mixed with Matrigel were injected around the implanted cells-BAM complex. In the results, compared with control rabbits received bladder augmentation with porcine BAM seeded with BSMCs, the experimental animals showed significantly improved smooth muscle regeneration and vascularization, along with more excellent functional recovery of tissue-engineered bladder, due to the additional combination of autologous EPCs and bioactive factors, including VEGF and PDGF-BB. Furthermore, cell tracking suggested that the seeded EPCs could be directly involved in neovascularization. Therefore, it may be an effective method to combine multiple pathways for tissue-engineering urinary bladder.

Bioprinting on sheet-based scaffolds applied to the creation of implantable tissue-engineered constructs with potentially diverse clinical applications: Tissue-Engineered Muscle Repair (TEMR) as a representative testbed

ABSTRACTPurpose: This report explores the overlooked potential of bioprinting to automate biomanufacturing of simple tissue structures, such as the uniform deposition of (mono)layers of progenitor cells on sheetlike decellularized extracellular matrices (dECM). In this scenario, dECM serves as a biodegradable celldelivery matrix to provide enhanced regenerative microenvironments for tissue repair. The Tissue-Engineered Muscle Repair (TEMR) technology—where muscle progenitor cells are seeded onto a porcine bladder acellular matrix (BAM), serves as a representative testbed for bioprinting applications. Previous work demonstrated that TEMR implantation improved functional outcomes following VML injury in biologically relevant rodent models.Materials and Methods: In the described bioprinting system, a cell-laden hydrogel bioink is used to deposit high cell densities (1.4 × 105-3.5 × 105 cells/cm2), onto both sides of the bladder acellular matrix as proof-of-concept.Results: These bioprinting methods achieve a reproducible and homogeneous distribution of cells, on both sides of the BAM scaffold, after just 24hrs, with cell viability as high as 98%. These preliminary results suggest bioprinting allows for improved dual-sided cell coverage compared to manual-seeding.Conclusions: Bioprinting can enable automated fabrication of TEMR constructs with high fidelity and scalability, while reducing biomanufacturing costs and timelines. Such bioprinting applications are underappreciated, yet critical, to expand the overall biomanufacturing paradigm for tissue engineered medical products. In addition, biofabrication of sheet-like implantable constructs, with cells deposited on both sides, is a process that is both scaffold and cell-type agnostic, and furthermore, is amenable to many geometries, and thus, additional tissue engineering applications beyond skeletal muscle.

Hypoxia-Preconditioned Adipose-Derived Endothelial Progenitor Cells Promote Bladder Augmentation.

Tissue engineering technology provides an alternative for bladder reconstruction, which remains confronted with challenges, such as insufficient vascularization in regenerated bladder tissue. Short-term hypoxic preconditioning has been reported to be an effective method to enhance the angiogenic effect of stem cells. This study evaluated the effect and mechanism of hypoxia-preconditioned adipose-derived endothelial progenitor cells (hp-ADEPCs) on the vascularization and smooth muscle regeneration of tissue-engineered bladders. Rats were randomly divided into the following four groups: hp-ADEPC, ADEPC, bladder acellular matrix (BAM), and cystotomy groups. A partial cystectomy was performed to remove 50% of the bladder, which was augmented with hp-ADEPC-BAM, ADEPC-BAM, or BAM. Histological and functional assessments of the engineered neobladder were performed at 1 and 3 months after surgery, while the mechanism of hp-ADEPCs on vascularization was also investigated. Immunohistochemical analysis revealed that hp-ADEPC-BAM significantly promoted urothelium, blood vessel, smooth muscle, and nerve cell regeneration. Animals in the hp-ADEPC-BAM group exhibited higher bladder compliance and a relatively normal micturition pattern compared with the ADEPC-BAM and BAM groups. In addition, compared with ADEPCs, hp-ADEPCs promoted ERK phosphorylation activation and hypoxia-inducible factor-1α expression, thereby secreting more vascular endothelial growth factor and basic fibroblast growth factor and significantly enhancing the migration and angiogenesis abilities of rat endothelial cells. This is the first study to demonstrate that a combination of ADEPCs and BAM with short-term hypoxic preconditioning enhances angiogenesis and promotes the functional recovery of tissue-engineered bladders. Impact Statement Insufficient vascularization in regenerated bladder tissue remains a challenge for bladder tissue engineering. We successfully isolated adipose-derived endothelial progenitor cells (ADEPCs) with high proliferative potential and angiogenic properties. Hypoxic preconditioning was confirmed to effectively enhance stem cell activity. In this study, a porcine bladder acellular matrix (BAM) was established, and hypoxia-preconditioned autologous ADEPCs were simultaneously introduced for bladder reconstruction in a rat model, followed by an assessment of their feasibility and potential for bladder vascularization. For the first time, we demonstrated that hypoxic preconditioning of ADEPCs improves angiogenesis and the functional recovery of tissue-engineered bladders.

Using Bioprinting to Tissue Engineer Microvascularized Constructs for Skeletal Muscle Repair

IntroductionOne challenge in tissue engineering (TE) is designing a construct which ensures adequate oxygen diffusion and removal of waste throughout the implanted tissue. In native tissue, the maximum distance between any cell and capillary is limited to the diffusion distance of oxygen, or 100–200 μm1. Mimicking this dense microvasculature in TE constructs is critical to cell viability and function, particularly in thick implants, such as those for skeletal muscle repair2. There have been many recent advancements in using TE to develop skeletal muscle constructs for the treatment of volumetric muscle loss (VML) in injuries incurred by soldiers and civilians. We have developed a tissue engineered muscle repair (TEMR) technology which consists of myoblasts seeded onto a porcine bladder acellular matrix (BAM). While TEMR has previously been reported to result in 60–70% functional recovery in rodent models within 2 months after implantation3,4, we hypothesize that incorporating endothelial cells (ECs) and pericytes in the TEMR will accelerate the rate and magnitude of functional recovery by facilitating anastomosis with the recipient microcirculation and increasing perfusion into the construct.ObjectiveUse bioprinting to position endothelial cells and pericytes with myoblasts on the BAM to “pre‐vascularize” the TEMR construct.ResultsDecellularized microvasculature basement membrane remains intact in some areas of the BAM (Figure 1A). Using the Organovo NovoGen Bioprinter®, we have developed a novel bioprinting method to pattern ECs and myoblasts on the acellular bladder matrix (BAM). ECs seeded onto the BAM begin to form network‐like structures after just 24 hours (Figure 1B). EC viability was shown to be high (>90%) for two bioprinting needle diameters (250 μm vs 500 μm). (Figure 1C).ConclusionsThe presence of ECM from microvasculature on the BAM suggests that the BAM is a promising scaffold for pre‐vascularization. This is further supported by the formation of endothelial network‐like formations on the BAM after just 24 hrs. It is also encouraging that the 250 μm bioprinting needle does not reduce cell viability, as a smaller diameter allows for more precise patterning of cells. Together, these preliminary results suggest that it is feasible to “pre‐vascularize” the TEMR construct by bioprinting.Future WorkWe will further investigate EC viability and network formation in co‐culture with myoblasts. We also plan to incorporate bioprinted pericytes to further encourage and support the formation of pre‐vascularized microvascular networks in TEMR.Support or Funding InformationNIH T32 GM008715, Organovo, Inc., and the UVA Center for Advanced BiomanufacturingThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

254 - Co-administration of PDGF-BB and VEGF with bladder acellular matrix enhance smooth muscle regeneration and vascularization for bladder augmentation in a rabbit model

Tissue engineering techniques has brought great hope for bladder repair and reconstruction. The crucial requirements of tissue engineered bladder are bladder smooth muscle regeneration and vascularization. In this study, partial rabbit bladder (4 cm×5 cm) was removed and replaced with porcine bladder acellular matrix (BAM) which was equal in size. BAM was incorporated with platelet-derived growth factor-BB (PDGF-BB) and vascular endothelial growth factor (VEGF) in the experimental group while with no bioactive factors in the control group. The bladder tissue strip contractility in the experimental rabbits was better than that in the control ones post-operation. Histological evaluation revealed that smooth muscle regeneration and vascularization in the experimental group were significantly improved compared with those in the control group (p < 0.05), while multilayered urothelium was formed in both groups. Muscle strip contractility of neo-bladder in the experimental exhibited significantly better than that in the control (p < 0.05) assessed with electrical field stimulation and carbachol interference. The activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) in the native bladder tissue around tissue engineered neo-bladder in the experimental group were significantly higher than those in the control (p < 0.05). This work suggests that smooth muscle regeneration and vascularization in tissue engineered neo-bladder and recovery of bladder function could be enhanced by PDGF-BB and VEGF incorporated within BAM, which promoted the up-regulation of the activity of MMP-2 and MMP-9 of native bladder tissue around the tissue engineered neo-bladder.

Autologous Smooth Muscle Progenitor Cells Enhance Regeneration of Tissue-Engineered Bladder.

Tissue engineering techniques provide a great potential to de novo construct a histological bladder. Smooth muscle regeneration is extremely important for the functional recovery of engineered neobladder. However, many challenges remain for the use of bladder smooth muscle cells (SMCs) as the cell sources. Recent evidences showed that smooth muscle progenitor cells (SPCs) in the peripheral blood have the capacity of differentiating into SMCs, while their use for bladder regeneration has not yet been reported. The aim of our study was to investigate the effect and mechanism of autologous SPCs on bladder regeneration in a rabbit model. In this study, autologous SPCs were isolated and cultured from the peripheral blood, labeled with CM-DiI, and then seeded into a porcine bladder acellular matrix (BAM) to construct a SPC-BAM complex, which was finally implanted to substitute the partial bladder with an equivalent size. In the results, SPCs demonstrated the phenotype of stem/progenitor cells, expressed SMs markers (alpha-smooth muscle actin [α-SMA], desmin, calponin, SM22α, and smooth muscle myosin heavy chain [SMMHC]), and displayed carbachol-induced contraction. Compared with cell-free BAM, the SPC-BAM was able to improve histological regeneration (smooth muscle regeneration, vascularization, and nerve formation) and functional recovery (urodynamic function and smooth muscle contraction) of the engineered neobladder. Cell tracing indicated that seeded SPCs could survive and directly integrated into the regenerated neobladder. In addition, SPCs could also promote proliferation and migration of rabbit bladder SMCs through the paracrine platelet-derived growth factor-BB (PDGF-BB). In conclusion, our study first demonstrated that SPCs from the peripheral blood could enhance histological regeneration and functional recovery of the tissue-engineered neobladder through both the direct integration and indirect paracrine effect, supporting the use of SPCs as the cell sources for tissue engineering of the bladder.

Coadministration of Platelet-Derived Growth Factor-BB and Vascular Endothelial Growth Factor with Bladder Acellular Matrix Enhances Smooth Muscle Regeneration and Vascularization for Bladder Augmentation in a Rabbit Model

Tissue-engineering techniques have brought a great hope for bladder repair and reconstruction. The crucial requirements of a tissue-engineered bladder are bladder smooth muscle regeneration and vascularization. In this study, partial rabbit bladder (4×5 cm) was removed and replaced with a porcine bladder acellular matrix (BAM) that was equal in size. BAM was incorporated with platelet-derived growth factor-BB (PDGF-BB) and vascular endothelial growth factor (VEGF) in the experimental group while with no bioactive factors in the control group. The bladder tissue strip contractility in the experimental rabbits was better than that in the control ones postoperation. Histological evaluation revealed that smooth muscle regeneration and vascularization in the experimental group were significantly improved compared with those in the control group (p<0.05), while multilayered urothelium was formed in both groups. Muscle strip contractility of neobladder in the experimental group exhibited significantly better than that in the control (p<0.05) assessed with electrical field stimulation and carbachol interference. The activity of matrix metalloproteinase-2 (MMP-2) and MMP-9 in the native bladder tissue around tissue-engineered neobladder in the experimental group was significantly higher than that in the control (p<0.05). This work suggests that smooth muscle regeneration and vascularization in tissue-engineered neobladder and recovery of bladder function could be enhanced by PDGF-BB and VEGF incorporated within BAM, which promoted the upregulation of the activity of MMP-2 and MMP-9 of native bladder tissue around the tissue-engineered neobladder.

Sustained Release of VEGF from PLGA Nanoparticles Embedded Thermo-sensitive Hydrogel in Full-thickness Porcine Bladder Acellular matrix

Sustained Release of VEGF from PLGA Nanoparticles Embedded Thermo-sensitive Hydrogel in Full-thickness Porcine Bladder Acellular matrix

A Tissue-Engineered Muscle Repair Construct for Functional Restoration of an Irrecoverable Muscle Injury in a Murine Model

There are no effective clinical treatments for volumetric muscle loss (VML) resulting from traumatic injury, tumor excision, or other degenerative diseases of skeletal muscle. The goal of this study was to develop and characterize a more clinically relevant tissue-engineered muscle repair (TE-MR) construct for functional restoration of a VML injury in the mouse lattissimus dorsi (LD) muscle. To this end, TE-MR constructs developed by seeding rat myoblasts on porcine bladder acellular matrix were preconditioned in a bioreactor for 1 week and implanted in nude mice at the site of a VML injury created by excising 50% of the native LD. Two months postinjury and implantation of TE-MR, maximal tetanic force was ∼72% of that observed in native LD muscle. In contrast, injured LD muscles that were not repaired, or were repaired with scaffold alone, produced only ∼50% of native LD muscle force after 2 months. Histological analyses of LD tissue retrieved 2 months after implantation demonstrated remodeling of the TE-MR construct as well as the presence of desmin-positive myofibers, blood vessels, and neurovascular bundles within the TE-MR construct. Overall, these encouraging initial observations document significant functional recovery within 2 months of implantation of TE-MR constructs and provide clear proof of concept for the applicability of this technology in a murine VML injury model.

Sustained release of VEGF from PLGA nanoparticles embedded thermo-sensitive hydrogel in full-thickness porcine bladder acellular matrix

We fabricated a novel vascular endothelial growth factor (VEGF)-loaded poly(lactic-co-glycolic acid) (PLGA)-nanoparticles (NPs)-embedded thermo-sensitive hydrogel in porcine bladder acellular matrix allograft (BAMA) system, which is designed for achieving a sustained release of VEGF protein, and embedding the protein carrier into the BAMA. We identified and optimized various formulations and process parameters to get the preferred particle size, entrapment, and polydispersibility of the VEGF-NPs, and incorporated the VEGF-NPs into the (poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (Pluronic®) F127 to achieve the preferred VEGF-NPs thermo-sensitive gel system. Then the thermal behavior of the system was proven by in vitro and in vivo study, and the kinetic-sustained release profile of the system embedded in porcine bladder acellular matrix was investigated. Results indicated that the bioactivity of the encapsulated VEGF released from the NPs was reserved, and the VEGF-NPs thermo-sensitive gel system can achieve sol-gel transmission successfully at appropriate temperature. Furthermore, the system can create a satisfactory tissue-compatible environment and an effective VEGF-sustained release approach. In conclusion, a novel VEGF-loaded PLGA NPs-embedded thermo-sensitive hydrogel in porcine BAMA system is successfully prepared, to provide a promising way for deficient bladder reconstruction therapy.

Development of a Porcine Bladder Acellular Matrix with Well-Preserved Extracellular Bioactive Factors for Tissue Engineering

In this study, we compared four decellularization protocols and finally developed an optimized one through which a porcine bladder acellular matrix (BAM) with well-preserved extracellular bioactive factors had been prepared. In this protocol, the intact bladder was treated with trypsin/ethylenediaminetetraacetic acid to remove the urothelium, then with hypotonic buffer and Triton X-100 in hypertonic buffer to remove the membranous and cytoplasmic materials, and finally with nuclease to degrade the cellular nuclear components. Bladder distention and mechanical agitation were simultaneously used to facilitate cell removal. Meanwhile, several preservative techniques, including limitation of wash time, supplement with inhibitors of proteinase, control of the pH value and temperature of the wash buffer, ethylene oxide sterilization, and lyophilization of the scaffold for storage, were used to protect the extracellular bioactive factors. This decellularization protocol had completely removed the cellular materials and well preserved the extracellular collagen, sulfated glycosaminoglycan (GAG), and bioactive factors. The preserved bioactive factors had a great potential of promoting the proliferation and migration of both human bladder smooth muscle cell and human umbilical vein endothelial cell. It was also found that the amount of two representative bioactive factors, platelet-derived growth factor BB and vascular endothelial growth factor, was positively correlated with the sulfated GAG content in the porcine BAM, implying that the amount of sulfated GAG might be a determinant for preservation of bioactive factors in the decellularized tissues. In conclusion, the porcine BAM with well-preserved extracellular bioactive factors might be a favorable scaffold for tissue engineering applications.

Bladder tissue engineering: Tissue regeneration and neovascularization of HA‐VEGF‐incorporated bladder acellular constructs in mouse and porcine animal models

Successful tissue engineering requires appropriate recellularization and vascularization. Herein, we assessed the regenerative and angiogenic effects of porcine bladder acellular matrix (ACM) incorporated with hyaluronic acid (HA) and vascular endothelial growth factor (VEGF) in mouse and porcine models. Prepared HA-ACMs were rehydrated in different concentrations of VEGF (1, 2, 3, 10, and 50 ng/g ACM). Grafts were implanted in mice peritoneum in situ for 1 week. Angiogenesis was quantified with CD31 and Factor VIII immunostaining using Simple PCI. Selected optimal VEGF concentration that induced maximum vascularization was then used in porcine bladder augmentation model. Implants were left in for 4 and 10 weeks. Three groups of six pigs each were implanted with ACM alone, HA-ACM, and HA-VEGF-ACM. Histological, immunohistochemical (Uroplakin III, alpha-SMA, Factor VIII), and immunofluorescence (CD31) analysis were performed to assess graft regenerative capacity and angiogenesis. In mouse model, statistically significant increase in microvascular density was demonstrated in the 2 ng/g ACM group. When this concentration was used in porcine model, recellularization increased significantly from weeks 4 to 10 in HA-VEGF-ACM, with progressive decrease in fibrosis. Significantly increased vascularization, coupled with increased urothelium and smooth muscle cell (SMC) regeneration, was observed in HA-VEGF grafts at week 10 in the center and periphery, compared with week 4. HA-VEGF grafts displayed highest in vivo epithelialization, neovascularization, and SMCs regeneration. A total of 2 ng/g tissue VEGF when incorporated with HA proved effective in stimulating robust graft recellularization and vascularization, coordinated with increased urothelial bladder development and SMC augmentation into bundles by week 10.

Bioengineered skeletal muscle for defect replacement in a rodent model

Loss of functional skeletal muscle due to congenital and acquired conditions, such as traumatic injury, tumor excision, etc., produces a physiological deficit for which there is still no effective clinical treatment. The goal of these studies is to develop clinically relevant tissue engineered skeletal muscle (TESKM) that can eventually be used for functional restoration of injured muscle in vivo. TESKM constructs developed by seeding rat muscle precursor cells on porcine bladder acellular matrix were preconditioned in a bioreactor for one week and implanted in nude mice (for 1–2 months) to repair a defect created by excising 50% of the native Latissimus Dorsi muscle (LD). Isometric tetanic specific force (ITSF) generated by the un‐repaired defect only group (49.2± 1.8 mN/mm2) was significantly lower (p&lt;0.01) than that of native LD (161.1± 22.4 mN/mm2). ITSF of TESKM constructs increased over time from one month (20.06± 6.4 mN/mm2) to two month (86.75± 45.7 mN/mm2) which is 53% of native LD. Besides this, the TESKM constructs recorded higher tension than the unrepaired group (12.6± 4.1 g) both at 1 month (16.88± 7.1 g) and 2 month (22.34± 5.6 g, p&lt;0.05), with the latter being 73% of native LD (30.67± 4.4g). Taken together, these initial observations indicate that TESKM can generate approximately half the ITSF of native LD after two months of implantation, illustrating the potential clinical utility of this approach.Grant Funding Source: AFIRM

Mouse Latissimus Dorsi as a model system for evaluating tissue engineered skeletal muscle

The inability to engineer clinically relevant functional muscle tissues in vitro remains a major obstacle to the development of successful procedures for skeletal muscle (SKM) replacement in vivo. The goal of these studies is to further develop the technologies required for the creation of skeletal muscle biomimetics that can be used clinically for restoration of muscle function in vivo. As a first step in this direction we have examined the rodent latissimus dorsi (LD) as a model system for testing functional replacement of native SKM with tissue engineered SKM constructs (TE‐SKM). Porcine bladder acellular matrix was prepared and rat muscle precursor cells were seeded onto the matrix and preconditioned in a custom designed, computer controlled cyclic strain bioreactor for one week prior to subcutaneous implantation in nu/nu mice onto LD for 1 month. The size of the TE‐SKM was 15 mm X 3mm X 0.35 mm; dimensions similar to that of the native mouse LD. TE‐SKM isolated after one month implantation showed a maximal isometric tetanic response of 42.9 mN/mm2 which was 26 % of that of native LD isolated from contra lateral side, but 50‐ and 3500‐fold greater than that previously observed for TE‐SKM. To our knowledge, this is the largest specific force yet recorded for TE‐SKM on a decellularized matrix. In short, the LD appears to be an attractive model for further studies of functional SKM replacement with TE‐SKM constructs.

Porcine bladder acellular matrix (ACM): protein expression, mechanical properties

Experimentally, porcine bladder acellular matrix (ACM) that mimics extracellular matrix has excellent potential as a bladder substitute. Herein we investigated the spatial localization and expression of different key cellular and extracellular proteins in the ACM; furthermore, we evaluated the inherent mechanical properties of the resultant ACM prior to implantation. Using a proprietary decellularization method, the DNA contents in both ACM and normal bladder were measured; in addition we used immunohistochemistry and western blots to quantify and localize the different cellular and extracellular components, and finally the mechanical testing was performed using a uniaxial mechanical testing machine. The mean DNA content in the ACM was significantly lower in the ACM compared to the bladder. Furthermore, the immunohistochemical and western blot analyses showed that collagen I and IV were preserved in the ACM, but possibly denatured collagen III in the ACM. Furthermore, elastin, laminin and fibronectin were mildly reduced in the ACM. Although the ACM did not exhibit nucleated cells, residual cellular components (actin, myosin, vimentin and others) were still present. There was, on the other hand, no significant difference in the mean stiffness between the ACM and the bladder. Although our decellularization method is effective in removing nuclear material from the bladder while maintaining its inherent mechanical properties, further work is mandatory to determine whether these residual DNA and cellular remnants would lead to any immune reaction, or if the mechanical properties of the ACM are preserved upon implantation and cellularization.

Porcine vesical acellular matrix graft of tunica albuginea for penile reconstruction

To characterize the feasibility of the surgical replacement of the penile tunica albuginea (TA) and to evaluate the value of a porcine bladder acellular matrix (BAM) graft. Acellular matrices were constructed from pigs' bladders by cell lysis, and then examined by scanning electron microscopy (SEM). Expression levels of the mRNA of the vascular endothelial growth factor (VEGF) receptor, fibroblast growth factor (FGF)-1 receptor, neuregulin, and brain-derived neurotrophic factor (BDNF) in the acellular matrix and submucosa of the pigs'bladders were determined through the reverse transcription-polymerase chain reaction (PCR). A 5 mm X 5 mm square was excised from the penile TA of nine rabbits. The defective TA was then covered in porcine BAM. Equal numbers of animals were sacrificed and histochemically examined at 2, 4 and 6 months after implantation. SEM of the BAM showed collagen fibers with many pores. VEGF receptor, FGF-1 receptor and neuregulin mRNA were expressed in the porcine BAM; BDNF mRNA was not detected. Two months after implantation, the graft sites exhibited excellent healing without contracture, and the fusion between the graft and the neighboring normal TA appeared to be well established. There were no significant histological differences between the implanted tunica and the normal control tunica at 6 months after implantation. The porcine BAM graft resulted in a structure which was sufficiently like that of the normal TA. This implantation might be considered applicable to the reconstruction of the TA in conditions such as trauma or Peyronie's disease.

Porcine bladder acellular matrix porosity: Impact of hyaluronic acid and lyophilization

Bladder acellular matrix (ACM) is being investigated as a urinary bladder replacement scaffold. We have demonstrated that ACM is porous and theorized that this contributes to ACM fibrosis and contracture over time in vivo and may preclude uptake and retention of molecules, which may aid cellular repopulation. We sought to determine if hyaluronic acid (HA) would decrease ACM porosity. Porcine ACM was lyophilized and rehydrated in HA (SIGMA) to form the hybrid HA-ACM construct. Three groups (n = 15/group: HA-ACM, ACM, and lyophilized/rehydrated ACM) were tested for porosity to a 10 cm column of distilled water, measuring the effluent hourly for 3 h. A porosity index was determined as the total effluent divided by time and area (cc/cm2 hr). Alcian blue staining and fluorophore-assisted carbohydrate electrophoresis qualitatively and quantitatively confirmed the uptake of HA. HA-ACM and lyophilized/rehydrated ACM were significantly less porous to water than untreated ACM [mean (+/-SE): 0.09 (+/-0.02), 0.74 (+/-0.4), and 9.8 (+/-1.6) cc/cm2 hr, respectively; Mann Whitney p < 0.0001 (HA) and p < 0.0001 (lyo)]. The difference between HA-ACM and lyophilized ACM was also statistically significant (p = 0.014). ACM hybridization with HA decreases ACM porosity, in part because of ACM lyophilization during the hybridization process. In future applications, HA may function as a carrier for smaller molecules such as growth factors, and as a bioactive molecule to improve wound healing and decrease fibrosis in tissue-engineered bladder constructs.

Preliminary research on preparation of porcine bladder acellular matrix graft for tissue engineering applications

To investigate the preparation method of porcine bladder acellular matrix graft (BAMG) and evaluate the feasibility of using BAMG as biomaterial scaffold to construct tissue engineering bladder. Bladders were harvested from adult pigs at a slaughterhouse and were decellularized by the method of enzyme digestion or eradicator washing. The BAMG was then examined by optic microscope and electron microscope to confirm no cell elements remained. The structure of the BAMG was always observed through the Scanning Microscope. The skeletal muscle stem cells of New Zealand white rabbits were implanted in 96-hole-plank and cultured by BAMG extract liquid or normal culture medium. The cytotoxicity of BAMG was tested through MTT assay. New Zealand white rabbits were underwent bladder triangle area spared cystectomy and reconstructed by the heterogeneity BAMG. The animal bladders were obtained after six weeks and the tissue regeneration was examined. Good outcome were obtained with either of the two methods. There were no cell elements remained under the examination of optical microscope and electron microscope. The interstice could observed on BAMG through the Scanning Microscope. The relative growth rate (RGR) was 81%, 88%, 93% and 96% separately on the 2nd, the 4th, the 6th and the 8th day (t = 3.7419, P < 0.025). The cytotoxicity score was I, which indicated that BAMG had a good cytocompatibility. All rabbits were alive except one which died from diarrhea. The reconstructed bladder resumed the construction and function of the bladder. The transitional cells and muscle cells growth could be seen in the scaffold of the repaired bladder after six weeks. The porcine BAMG has good biocompatibility and can be used as ideal biomaterial scaffold of bladder tissue engineering.

Porosity of porcine bladder acellular matrix: impact of ACM thickness.

The objectives of this study are to examine the porosity of bladder acellular matrix (ACM) using deionized (DI) water as the model fluid and dextran as the indicator macromolecule, and to correlate the porosity to the ACM thickness. Porcine urinary bladders from pigs weighing 20-50 kg were sequentially extracted in detergent containing solutions, and to modify the ACM thickness, stretched bladders were acellularized in the same manner. Luminal and abluminal ACM specimens were subjected to fixed static DI water pressure (10 cm); and water passing through the specimens was collected at specific time interval. While for the macromolecule porosity testing, the diffusion rate and direction of 10,000 MW fluoroescein-labeled dextrans across the ACM specimens mounted in Ussing's chambers were measured. Both experiments were repeated on the thin stretched ACM. In both ACM types, the fluid porosity in both directions did not decrease with increased test duration (3 h); in addition, the abluminal surface was more porous to fluid than the luminal surface. On the other hand, when comparing thin to thick ACM, the porosity in either direction was higher in the thick ACM. Macromolecule porosity, as measured by absorbance, was higher for the abluminal thick ACM than the luminal side, but this characteristic was reversed in the thin ACM. Comparing thin to thick ACM, the luminal side in the thin ACM was more porous to dextran than in the thick ACM, but this characteristic was reversed for the abluminal side. The porcine bladder ACM possesses directional porosity and acellularizing stretched urinary bladders may increase structural density and alter fluid and macromolecule porosity.