Autologous Extracellular Matrix Research Articles

Abstract 3076: Autologous ECM-composed scaffolds and zebrafish PDXs for individualized theranostics in rare neuroendocrine neoplasms

Abstract Background: Neuroendocrine neoplasms (NENs) are a subgroup of poorly characterized rare tumors with challenging management. Identification of clinically useful biomarkers for patient stratification and treatment tailoring is urgently needed. This study proposes the use of near-patient models based on autologous extracellular matrix (aECM)-scaffolds and zebrafish (ZF) xenografts integrated with next generation multi-omics analysis for the phenotype and genotype mapping of NEN cells. The aim is to uncover molecular signatures associated with disease aggressiveness and to detect oncogenic drivers for targeted treatment. Methods: Primary tumor cells isolated from surgical specimens of NEN patients were cultured in aECM scaffolds or injected into ZF embryos. Autologous ECM-scaffolds were synthesized starting from the patients’ matrices and recellularized with primary cells derived from matched patients. In aECM scaffolds we evaluated tumor cell proliferation and neuroendocrine differentiation. In ZF we evaluated cell angiogenic ability and distant invasiveness. For each primary culture, we performed exome and transcriptome analysis of the original tumor tissue compared to the matched healthy counterpart. Results: We demonstrated that ZF xenografting faithfully recreates the behavior that tumor cells exhibit in patients. Specifically, we derived a primary culture from an ileal grade 1 neuroendocrine tumor from both the primary and the metastatic lesions. When injected into ZF embryos, cells derived from the primary tumor displayed low invasive ability with a metastatic rate of only 23%. Conversely, cells derived from the metastatic lesion were able to invade the ZF circulation and form distant metastases in 79% of embryos. Consecutively, we derived three primary cultures of neuroendocrine Merkel cell carcinoma (MCC). The 3 MCC cultures into ZF embryos showed high aggressiveness with metastatic rates of 81%, 50% and 64%. For one culture, angiogenic ability was also observed. RNA-sequencing of these aggressive MCC specimens highlighted the involvement of the following pathways: ECM remodeling, TNF-ⲁ and PI3K-Akt signaling, epithelial-mesenchymal transition and regulation of apoptotic processes. Next, we demonstrated that aECM-scaffolds morphologically resemble a native tumor tissue, and tumor cells, once seeded on them, preserved the expression of neuroendocrine markers. Conclusions: Here we developed innovative neuroendocrine tumor models that are expected to generate new knowledge on the disease molecular determinants. All identified genomic and genetic alterations will be correlated with the clinical outcomes of enrolled patients, to validate their clinical significance and to ultimately introduce a biomarker-based approach for NEN patients. Citation Format: Chiara Calabrese, Giacomo Miserocchi, Silvia Vanni, Alessandro De Vita, Alberto Bongiovanni, Chiara Spadazzi, Claudia Cocchi, Nicoletta Ranallo, Federica Pieri, Michela Tebaldi, Flavia Foca, Giorgio Ercolani, Davide Cavaliere, Carlo Alberto Pacilio, Giovanni Martinelli, Laura Mercatali, Chiara Liverani. Autologous ECM-composed scaffolds and zebrafish PDXs for individualized theranostics in rare neuroendocrine neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3076.

84P Autologous ECM-composed scaffolds and zebrafish PDXs for individualized theranostics in rare neuroendocrine neoplasms

Neuroendocrine neoplasms (NENs) are a subgroup of poorly characterized rare tumors with challenging management. NENs are very heterogeneous and the identification of clinically useful biomarkers for patient stratification and treatment tailoring is urgently needed. This study proposes the use of near-patient models based on autologous extracellular matrix (aECM)-scaffolds and zebrafish (ZF) xenografts coupled with next generation sequencing techniques for the phenotype and genotype mapping of primary NEN cells.

2155-PUB: Diabetic Foot Ulcer Regeneration Platform Based on 4D Bioprinting Technology

Diabetic foot ulcers have a significantly negative impact on patients and are highly prone to contamination, which can lead to amputation. The best possible wound management treatment was essential for diabetic foot ulcers. However, since 4D bio printing is the best way to recapitulate complex and functional human tissue and organ, we have developed the therapeutic 4D bio printer called INVIVO to use in skin regeneration. To find advanced treatment method particularly for wound healing, we tested our own 4D bio printing system with autologous fat tissue for DFU treatment. The extracellular matrix (ECM) derived from Nano-fat consisting of supportive proteins, growth factors and cytokines, has been reported as an important player to provide efficient 3D environments during cell proliferation and differentiation. In the clinical study, we printed autologous ECM (MA-ECM) with bio-inks to apply onto the chronic wound site. Most of DFU patients showed significant reduction of wound size with distinct epithelization only after a week treatment. Therefore, we suggest 4D bio printing system with MA-ECM as an alternative method for dressing materials that successfully promote the mechanism of skin reconstruction for DFU treatment. Reduction in size of wound was measured using ImageJ NIH software and almost all the subjects revealed complete closure of wound maximum of 2∼5 weeks post MA-ECM membrane application. The present study provided a significantly higher healing rate of the subjects and the primary outcome of our study complete epithelization with wound closure was achieved. The autologous fat by nature is loaded with adipocytes and growth factors which could have played a vital role in the healing of non-healing diabetic wounds. Therefore, we suggest 4D bio printing system with MA-ECM as an alternative method for chronic wound healing that successfully promote the mechanism of skin reconstruction for DFU treatment. Disclosure J. Kim: None.

Subcutaneously engineered autologous extracellular matrix scaffolds with aligned microchannels for enhanced tendon regeneration: Aligned microchannel scaffolds for tendon repair

Improved strategies for the treatment of tendon defects are required to successfully restore mechanical function and strength to the damaged tissue. This remains a scientific and clinical challenge, given the tendon's limited innate regenerative capacity. Here, we present an engineering solution that stimulates the host cell's remodeling abilities. We combined precision-designed templates with subcutaneous implantation to generate decellularized autologous extracellular matrix (aECM) scaffolds that had highly aligned microchannels after removal of templates and cellular components. The aECM scaffolds promoted rapid cell infiltration, favorable macrophage responses, collagen-rich extracellular matrix (ECM) synthesis, and physiological tissue remodeling in rat Achilles tendon defects. At three months post-surgery, the mechanical strength of tenocyte-populated ‘neo-tendons' was comparable to pre-injury state tendons. Overall, we demonstrated an in vivo bioengineering strategy for improved restoration of tendon tissue, which also offers wider implications for the regeneration of other highly organized tissues.

Engineering of Human Skeletal Muscle With an Autologous Deposited Extracellular Matrix.

Adult skeletal muscle progenitor cells can be embedded in an extracellular matrix (ECM) and tissue-engineered to form bio-artificial muscles (BAMs), composed of aligned post-mitotic myofibers. The ECM proteins which have been used most commonly are collagen type I and fibrin. Fibrin allows for in vitro vasculogenesis, however, high concentrations of fibrinolysis inhibitors are needed to inhibit degradation of the ECM and subsequent loss of BAM tissue structure. For in vivo implantation, fibrinolysis inhibition may prove difficult or even harmful to the host. Therefore, we adapted in vitro culture conditions to enhance the deposition of de novo synthesized collagen type I gradually replacing the degrading fibrin ECM. The in vitro viscoelastic properties of the fibrin BAMs and deposition of collagen were characterized. BAMs engineered with the addition of proline, hydroxyproline, and ascorbic acid in the tissue culture medium had a twofold increase in Young’s Modulus, a 2.5-fold decrease in maximum strain, and a 1.6-fold increase in collagen deposition. Lowering the fibrin content of the BAMs also increased Young’s Modulus, decreased maximum strain, and increased collagen deposition. Tissue engineering of BAMs with autologous ECM may allow for prolonged in vivo survival.

Results of treatment of scaffold-free pellet-type autologous chondrocytes implantation (cartilifeTM) in patients with knee cartilage lesions. a multi-center, active-controlled, randomized trial

Purpose: To compare the 1-year follow up results of the structural regeneration of the cartilage lesion and clinical outcome of patients treated using CartiLifeTM with those treated using microfracture (MFx). Methods: Thirty patients with an ICRS grade 3 or 4 chondral defect (2 to 10 cm2 in area; ≤ 4 cm3 in volume) were randomized at a ratio of two (CartiLifeTM) to one (MFx). CartiLifeTM is a scaffold-free bead-type ACI product composed of autologous extracellular matrix and chondrocytes. The implantation of CartiLifeTM was performed through minimal arthrotomy. The small beads of CartiLifeTM were implanted in the defect to the level of adjacent cartilage and then fixed with fibrin glue. MRI MOCART scores were used for structural assessment and Lysholm, IKDC, KOOS and VAS scores for clinical assessment 24 and 48 weeks post-operation. Results: The MOCART scores improved from the baseline to 24 and 48 weeks postoperatively in both treatment groups. Improvement for MOCART scores in the CartiLifeTM group was significantly greater than that of the MFx group at 24 and 48 weeks (p = 0.0052 and p = 0.0024, respectively). The Lysholm, IKDC, KOOS and VAS scores significantly improved from baseline to 24 and 48 weeks in both treatment groups. Improvement of the Lysholm scores in the CartiLifeTM group was significantly greater than that of the MFx group at 48 weeks (p = 0.0057). Conclusions: The overall results of the randomized clinical trial indicated that treatment with CartiLifeTM can be a critical option for structural cartilage regeneration as well as improvement of patient symptoms and function. However, a long-term follow up study in a large number of patients should be considered.

Regeneration of Cartilage in Human Knee Osteoarthritis with Autologous Adipose Tissue-Derived Stem Cells and Autologous Extracellular Matrix.

This clinical case series demonstrates that percutaneous injections of autologous adipose tissue-derived stem cells (ADSCs) and homogenized extracellular matrix (ECM) in the form of adipose stromal vascular fraction (SVF), along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, could regenerate cartilage-like tissue in human knee osteoarthritis (OA) patients. Autologous lipoaspirates were obtained from adipose tissue of the abdominal origin. Afterward, the lipoaspirates were minced to homogenize the ECM. These homogenized lipoaspirates were then mixed with collagenase and incubated. The resulting mixture of ADSCs and ECM in the form of SVF was injected, along with HA and PRP activated by calcium chloride, into knees of three Korean patients with OA. The same affected knees were reinjected weekly with additional PRP activated by calcium chloride for 3 weeks. Pretreatment and post-treatment magnetic resonance imaging (MRI) data, functional rating index, range of motion (ROM), and pain score data were then analyzed. All patients' MRI data showed cartilage-like tissue regeneration. Along with MRI evidence, the measured physical therapy outcomes in terms of ROM, subjective pain, and functional status were all improved. This study demonstrates that percutaneous injection of ADSCs with ECM contained in autologous adipose SVF, in conjunction with HA and PRP activated by calcium chloride, is a safe and potentially effective minimally invasive therapy for OA of human knees.

Bioengineering vascularized tissue constructs using an injectable cell-laden enzymatically crosslinked collagen hydrogel derived from dermal extracellular matrix

Tissue engineering promises to restore or replace diseased or damaged tissue by creating functional and transplantable artificial tissues. The development of artificial tissues with large dimensions that exceed the diffusion limitation will require nutrients and oxygen to be delivered via perfusion instead of diffusion alone over a short time period. One approach to perfusion is to vascularize engineered tissues, creating a de novo three-dimensional (3D) microvascular network within the tissue construct. This significantly shortens the time of in vivo anastomosis, perfusion and graft integration with the host. In this study, we aimed to develop injectable allogeneic collagen-phenolic hydroxyl (collagen-Ph) hydrogels that are capable of controlling a wide range of physicochemical properties, including stiffness, water absorption and degradability. We tested whether collagen-Ph hydrogels could support the formation of vascularized engineered tissue graft by human blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSC) in vivo. First, we studied the growth of adherent ECFCs and MSCs on or in the hydrogels. To examine the potential formation of functional vascular networks in vivo, a liquid pre-polymer solution of collagen-Ph containing human ECFCs and MSCs, horseradish peroxidase and hydrogen peroxide was injected into the subcutaneous space or abdominal muscle defect of an immunodeficient mouse before gelation, to form a 3D cell-laden polymerized construct. These results showed that extensive human ECFC-lined vascular networks can be generated within 7days, the engineered vascular density inside collagen-Ph hydrogel constructs can be manipulated through refinable mechanical properties and proteolytic degradability, and these networks can form functional anastomoses with the existing vasculature to further support the survival of host muscle tissues. Finally, optimized conditions of the cell-laden collagen-Ph hydrogel resulted in not only improving the long-term differentiation of transplanted MSCs into mineralized osteoblasts, but the collagen-Ph hydrogel also improved an increased of adipocytes within the vascularized bioengineered tissue in a mouse after 1month of implantation. Statement of SignificanceWe reported a method for preparing autologous extracellular matrix scaffolds, murine collagen-Ph hydrogels, and demonstrated its suitability for use in supporting human progenitor cell-based formation of 3D vascular networks in vitro and in vivo. Results showed extensive human vascular networks can be generated within 7days, engineered vascular density inside collagen-Ph constructs can be manipulated through refinable mechanical properties and proteolytic degradability, and these networks can form functional anastomoses with existing vasculature to further support the survival of host muscle tissues. Moreover, optimized conditions of cell-laden collagen-Ph hydrogel resulted in not only improving the long-term differentiation of transplanted MSCs into mineralized osteoblasts, but the collagen-Ph hydrogel also improved an increased of adipocytes within the vascularized bioengineered tissue in a mouse.

Preliminary Report of In Vitro Reconstruction of a Vascularized Tendonlike Structure

A greater supply of tendinous tissue can be obtained through tissue engineering technology with increasing application of adult stem cells. It is well known that adipose-derived stem cells (ADSCs), found in abundance in adipose tissue, have the same differentiating capacity as mesenchymal stem cells yet have the advantage of being easily isolated. In the present study, we combined the great facility of ADSCs to differentiate with the application of an external mechanical stimulus to successfully create an in vitro reconstructed tendonlike structure with a microcapillary network. Hyalonect meshes (Fidia Advanced Biopolymers, Abano Terme, Padova, Italy) were used as scaffold. Human ADSCs were seeded onto the biomaterials, and the cell/scaffold constructs were cultured under mechanical stress for up to 15 days. Human tenocytes were used in the same conditions as control. Performance was assessed by histology, immunochemistry, ultrastructure, and biomolecular analysis. Adipose-derived stem cells seeded onto Hyalonect adhered and differentiated along the entire surface of the biomaterial and began to infiltrate within its structure. Subsequently, endothelial cells migrated, forming a capillary in the new extracellular matrix. This technique allowed for the creation of a vascularized tendon equivalent that could easily be detached from the bioreactor, thus facilitating its implant at the lesion site. These results highlight the biologic performance of biodegradable hyaluronic acid-based (HYAFF-11) scaffolds, which were shown to be suitable for deposition of the autologous extracellular matrix critical for ADSCs differentiation.

Feasibility of Autologous Bone Marrow Mesenchymal Stem Cell–Derived Extracellular Matrix Scaffold for Cartilage Tissue Engineering

Extracellular matrix (ECM) materials are widely used in cartilage tissue engineering. However, the current ECM materials are unsatisfactory for clinical practice as most of them are derived from allogenous or xenogenous tissue. This study was designed to develop a novel autologous ECM scaffold for cartilage tissue engineering. The autologous bone marrow mesenchymal stem cell-derived ECM (aBMSC-dECM) membrane was collected and fabricated into a three-dimensional porous scaffold via cross-linking and freeze-drying techniques. Articular chondrocytes were seeded into the aBMSC-dECM scaffold and atelocollagen scaffold, respectively. An in vitro culture and an in vivo implantation in nude mice model were performed to evaluate the influence on engineered cartilage. The current results showed that the aBMSC-dECM scaffold had a good microstructure and biocompatibility. After 4 weeks in vitro culture, the engineered cartilage in the aBMSC-dECM scaffold group formed thicker cartilage tissue with more homogeneous structure and higher expressions of cartilaginous gene and protein compared with the atelocollagen scaffold group. Furthermore, the engineered cartilage based on the aBMSC-dECM scaffold showed better cartilage formation in terms of volume and homogeneity, cartilage matrix content, and compressive modulus after 3 weeks in vivo implantation. These results indicated that the aBMSC-dECM scaffold could be a successful novel candidate scaffold for cartilage tissue engineering.

Vitalize synthetic vascular grafts in vivo

larity producing an autologous extra-cellular matrix, replacing the degrading (65% MW reduction) PCL scaffold (11.3±4.0 vs. 31.4±9.2; Pb.0001). Morphometry and SEM showed 100% neoendothelialisation for both grafts. Intimal hyperplasia thickness, length and area were higher in ePTFE compared to PCL. Conclusions: Synthetic, biodegradable small-calibre nano-fibre polycaprolactone grafts show better patency, compliance, endothelialisation, less intimal hyperplasia and calcification compared to the clinically used ePTFE grafts after long-term implantation in the rat aorta. Thus, such novel in situ tissue engineered grafts could become a promising option for cardiovascular revascularisation procedures.

Experimental noninferiority trial of synthetic small-caliber biodegradable versus stable vascular grafts

Long-term evolution of polycaprolactone vascular prostheses has been investigated recently. The goal of this study was to evidence a noninferiority of such grafts compared with expanded polytetrafluoroethylene (ePTFE) implants in an aortic replacement model in the rat. Fourteen anesthetized Sprague-Dawley rats received an infrarenal aortic graft (biodegradable, n = 8; expanded polytetrafluoroethylene, n = 6) replacement (end to end; inner diameter, 2 mm). Biodegradable grafts (polycaprolactone) were produced by random micro-/nanofiber electrospinning. After a median survival of 16.5 months, in vivo ultrasonography and angiography as well as postexplantation microcomputed tomography, histomorphometry, immunohistochemistry, and scanning electron microscopy were performed. Patency was 100% for polycaprolactone and 67% for ePTFE. No aneurysmal dilatation or stenoses were found in either group. Compliance was significantly higher for polycaprolactone compared with ePTFE (8.2 ± 1.0%/100 mm Hg vs 5.7 ± 0.7%/100 mm Hg; P < .01), but markedly reduced compared with adjacent native aortas and the control group. Histologically, low cellular in-growth was found in ePTFE whereas polycaprolactone showed significantly greater homogenous cellularity, producing an autologous extracellular matrix (10.8% ± 4.0% vs 32.1% ± 9.2%, P < .0001). Morphometry showed 100% neo-endothelialization for both grafts with a totally confluent endothelial coverage for polycaprolactone grafts by scanning electron microscope. More intimal hyperplasia was found in ePTFE compared with polycaprolactone grafts. Calcification was higher in ePTFE than in polycaprolactone grafts (15.8% vs 7.0%, P = .04) and was absent in controls. Outcomes of synthetic biodegradable nanofiber polycaprolactone grafts are not inferior compared with the clinically used expanded polytetrafluoroethylene grafts after long-term implantation in the rat aorta. Moreover, these implants show better patency, compliance, endothelialization, and cell in-growth, and less intimal hyperplasia and calcification than their counterparts.

Spherical indentation of free-standing acellular extracellular matrix membranes

Numerous scaffold materials have been developed for tissue engineering and regenerative medicine applications to replace or repair damaged tissues and organs. Naturally occurring scaffold materials derived from acellular xenogeneic and autologous extracellular matrix (ECM) are currently in clinical use. These biological scaffold materials possess inherent variations in mechanical properties. Spherical indentation or ball burst testing has commonly been used to evaluate ECM and harvested tissue due to its ease of use and simulation of physiological biaxial loading, but has been limited by complex material deformation profiles. An analytical methodology has been developed and applied to experimental load–deflection data of a model hyperelastic material and lyophilized ECM scaffolds. An optimum rehydration protocol was developed based on water absorption, hydration relaxation and dynamic mechanical analysis. The analytical methodology was compared with finite element simulations of the tests and excellent correlation was seen between the computed biaxial stress resultants and geometry deformations. A minimum rehydration period of 5min at 37°C was sufficient for the evaluated multilaminated ECM materials. The proposed approach may be implemented for convenient comparative analysis of ECM materials and source tissues, process optimization or during lot release testing.

Establishment of Clinically Compliant Human Embryonic Stem Cells in an Autologous Feeder-Free System

Applications of human embryonic stem cells (hESCs) are limited by the use of mouse embryonic fibroblasts feeder and animal-derived components during culture. In this study, we demonstrated the potential use of extracellular matrix (ECM) derived from the autologous feeders to support long-term undifferentiated growth of hESCs in xeno-free, serum-free, and feeder-free conditions. Autologous H9 ebF (feeder cells derived from outgrowth of embryoid body [EB] predifferentiated from H9 hESCs) was derived from EBs predifferentiated from H9 hESCs through a direct-plating outgrowth system. The ECM comprising collagen VI, laminin, and fibronectin was extracted from H9 ebF through a freeze-thaw procedure. The autologous ECM together with animal component-free TeSR™2 medium was used to support long-term growth of H1 and H9 hESC lines for up to 20 passages. The maintenance of hESC undifferentiated state by autologous ECM was confirmed by the positive staining of hESC-specific markers (Oct4, SSEA-4, and Tra-1-60) and the expression of pluripotency marker genes (Oct4, Nanog, and Sox2). Flow cytometry further showed that more than 99% of hESCs retained the expression of SSEA-3/4 after long-term culture on autologous ECM. Pluripotency of hESCs on ECM was further proven by in vitro EB formation and in vivo teratoma assay. Overall, this study suggested a strategy for efficient propagation of clinically compliant hESCs in an autologous feeder-free culture system.

Autologous extracellular matrix scaffolds for tissue engineering

Development of autologous scaffolds has been highly desired for implantation without eliciting adverse inflammatory and immune responses. However, it has been difficult to obtain autologous scaffolds by tissue decellularization because of the restricted availability of autologous donor tissues from a patient. Here we report a method to prepare autologous extracellular matrix (aECM) scaffolds by combining culture of autologous cells in a three-dimensional template, decellularization, and template removal. The aECM scaffolds showed excellent biocompatibility when implanted. We anticipate that “Full Autologous Tissue Engineering” can be realized to minimize undesirable host tissue responses by culturing the patient’s own cells in an aECM scaffold.

Use of macroporous gelatine spheres as a biodegradable scaffold for guided tissue regeneration of healthy dermis in humans: An in vivo study

If a biodegradable scaffold is applied, the dermis can be regenerated by guided tissue regeneration. Scaffolds can stimulate in-growth of cells from the surroundings that migrate into them and start to produce autologous extracellular matrix as the scaffold is degraded. Several materials are available, but most of them are in the form of sheets and need to be laid on an open wound surface. A number of injectable fillers have been developed to correct soft-tissue defects. However, none of these has been used for guided tissue regeneration. We present a new technique that could possibly be used to correct dermal defects by using macroporous gelatine spheres as a biodegradable scaffold for guided tissue regeneration. In eight healthy volunteers, intradermal injections of macroporous gelatine spheres were compared with injections of saline and hyaluronic acid (Restylane). Full-thickness skin biopsy specimens of the implants and surrounding tissue were removed 2, 8, 12 and 26 weeks after injection, and the (immuno)histological results were analysed. The Restylane merely occupied space. It shattered the dermal tissue and compressed collagen fibres and cells at the interface between the implant and the dermis. No regeneration of tissue was found with this material at any time. The macroporous gelatine spheres were populated with fibroblasts already after 2 weeks. After 8 weeks the spheres were completely populated by fibroblasts producing dermal tissue. After 12 and 26 weeks, the gelatine spheres had been more or less completely resorbed and replaced by vascularised neodermis. There were no signs of capsular formation, rejection or adverse events in any subject. Further in vivo studies in humans are needed to evaluate the effect of the macroporous spheres fully as a matrix for guided tissue regeneration with and without cellular pre-seeding. However, the results of this study indicate the possibility of using macroporous gelatine spheres as an injectable, three-dimensional, degradable matrix for guided tissue regeneration.

Evidence forIn VivoGrowth Potential and Vascular Remodeling of Tissue-Engineered Artery

Nondegradable synthetic polymer vascular grafts currently used in cardiovascular surgery have no growth potential. Tissue-engineered vascular grafts (TEVGs) may solve this problem. In this study, we developed a TEVG using autologous bone marrow-derived cells (BMCs) and decellularized tissue matrices, and tested whether the TEVGs exhibit growth potential and vascular remodeling in vivo. Vascular smooth muscle-like cells and endothelial-like cells were differentiated from bone marrow mononuclear cells in vitro. TEVGs were fabricated by seeding these cells onto decellularized porcine abdominal aortas and implanted into the abdominal aortas of 4-month-old, bone marrow donor pigs (n = 4). Eighteen weeks after implantation, the dimensions of TEVGs were measured and compared with those of native abdominal aortas. Expression of molecules associated with vascular remodeling was examined with reverse transcription-polymerase chain reaction assay and immunohistochemistry. Eighteen weeks after implantation, all TEVGs were patent with no sign of thrombus formation, dilatation, or stenosis. Histological and immunohistochemical analyses of the retrieved TEVGs revealed regeneration of endothelium and smooth muscle and the presence of collagen and elastin. The outer diameter of three of the four TEVGs increased in proportion to increases in body weight and outer native aorta diameter. Considerable extents of expression of molecules associated with extracellular matrix (ECM) degradation (i.e., matrix metalloproteinase and tissue inhibitor of matrix metalloproteinase) and ECM precursors (i.e., procollagen I, procollagen III, and tropoelastin) occurred in the TEVGs, indicating vascular remodeling associated with degradation of exogenous ECMs (implanted decellularized matrices) and synthesis of autologous ECMs. This study demonstrates that the TEVGs with autologous BMCs and decellularized tissue matrices exhibit growth potential and vascular remodeling in vivo of tissue-engineered artery.

Persistence of Collagen Type II Synthesis and Secretion in Rapidly Proliferating Human Articular ChondrocytesIn Vitro

Articular chondrocytes (AC) expanded in vitro for tissue engineering rapidly turn off collagen type II (COL2) synthesis. We wanted to inhibit this process sufficiently to obtain therapeutically useful numbers of AC without losing COL2 synthesis. To this end, AC were expanded on their own extracellular matrix (ECM) in structures designated chondrocytes in autologous ECM (CA-ECM). Here, AC maintained a rounded shape and proliferated rapidly. After 13-15 days in culture, 40 x 10(6) cells (median) could be obtained from a cartilage biopsy. Real-time RT-PCR showed a reduced, but persistent, production of COL2A1 mRNA at this time. Flow cytometry showed high levels of intracellular COL2, and immunogold electron microscopy showed high density of well-organized COL2 fibrils in newly synthesized ECM. Interestingly, high levels of COL1A1 mRNA and intracellular protein were detected, but no COL1 was found in the ECM. The slow loss of COL2A1 mRNA was paralleled by a loss of the COL2 regulating transcription factor SOX9 mRNA. Chromatin immunoprecipitation assays could not identify epigenetic histone modifications that would explain the observed changes in COL2 synthesis. Thus, the CA-ECM strategy allows AC to proliferate to clinically useful numbers while maintaining COL2 synthesis and secretion. This strategy may improve tissue engineering of joint surfaces.

Scaffold precoating with human autologous extracellular matrix for improved cell attachment in cardiovascular tissue engineering.

Cell attachment to a scaffold is a precondition for the development of bioengineered valves and vascular substitutes. This attachment is generally facilitated by the use of precoating factors, but some can cause toxic or immunologic side effects. Autologous extracellular matrix (ECM) is used as a precoating factor in our study. Ascending aortic tissue was cultured to obtain human myofibroblasts. Autologous ECM was extracted from the same aortic tissue. Poly(glycolic acid) (PGA) scaffolds were precoated with autologous ECM, human serum, or poly-L-lysine; the control group was pretreated with phosphate buffered saline (PBS). Myofibroblasts were seeded onto each scaffold, and the cell attachment was assayed and compared. Compared with the control group, precoating with human serum, poly-L-lysine, and ECM increased number of attached cells by 24%, 53%, and 48%, respectively. Differences between precoating groups were significant (p < 0.01), except for ECM versus poly-L-lysine. Scanning electron microscopy also demonstrated the high degree of cell attachment to the PGA fibers on scaffolds precoated with ECM and poly-L-lysine. Precoating polymeric scaffold with autologous human extracellular matrix is a very effective method of improving cell attachment in cardiovascular tissue engineering without the potential risk of immunologic reactions.

Immunohistochemical localization of extracellular matrix proteins in human glioma, both in vivo and in vitro.

Expression of type IV collagen, fibronectin and laminin in various types of primary human brain tumor sections and normal brain tissue sections as well as cultured glioma cell lines was examined by an immunofluorescence technique. Type IV collagen, fibronectin, and laminin were mainly localized to the basement membrane of the vasculature in glioblastoma, anaplastic astrocytoma, low grade glioma, and in normal brain. However, positive staining for all the extracellular matrix (ECM) components tested was found only in glioblastoma sections both in the cells and in the ECM. In all other tumor types and in normal brain tissue, the cells did not stain for any of the ECM components. Four glioblastoma cell lines and autologous ECM synthesized by respective glioblastoma cell lines also showed positive staining for type IV collagen, fibronectin and laminin in vitro. These results suggest that glioblastoma cells both in vitro and in vivo express the extracellular matrix components that are involved in the regulation of tumor cell invasion.