Cardiac Repair With a Novel Population of Mesenchymal Stem Cells Resident in the Human Heart.

Cardiac resident stem cells (CRSCs) hold much promise to treat heart disease but this remains a controversial field. Here, we describe a novel population of CRSCs, which are positive for W8B2 antigen and were obtained from adult human atrial appendages. W8B2(+) CRSCs exhibit a spindle-shaped morphology, are clonogenic and capable of self-renewal. W8B2(+) CRSCs show high expression of mesenchymal but not hematopoietic nor endothelial markers. W8B2(+) CRSCs expressed GATA4, HAND2, and TBX5, but not C-KIT, SCA-1, NKX2.5, PDGFRα, ISL1, or WT1. W8B2(+) CRSCs can differentiate into cardiovascular lineages and secrete a range of cytokines implicated in angiogenesis, chemotaxis, inflammation, extracellular matrix remodeling, cell growth, and survival. In vitro, conditioned medium collected from W8B2(+) CRSCs displayed prosurvival, proangiogenic, and promigratory effects on endothelial cells, superior to that of other adult stem cells tested, and additionally promoted survival and proliferation of neonatal rat cardiomyocytes. Intramyocardial transplantation of human W8B2(+) CRSCs into immunocompromised rats 1 week after myocardial infarction markedly improved cardiac function (∼40% improvement in ejection fraction) and reduced fibrotic scar tissue 4 weeks after infarction. Hearts treated with W8B2(+) CRSCs showed less adverse remodeling of the left ventricle, a greater number of proliferating cardiomyocytes (Ki67(+) cTnT(+) cells) in the remote region, higher myocardial vascular density, and greater infiltration of CD163(+) cells (a marker for M2 macrophages) into the border zone and scar regions. In summary, W8B2(+) CRSCs are distinct from currently known CRSCs found in human hearts, and as such may be an ideal cell source to repair myocardial damage after infarction.

Hair transplantation in mice: Challenges and solutions.

Hair follicle cells contribute to wound healing, skin circulation, and skin diseases including skin cancer, and hair transplantation is a useful technique to study the participation of hair follicle cells in skin homeostasis and wound healing. Although hair follicle transplantation is a well-established human hair-restoration procedure, follicular transplantation techniques in animals have a number of shortcomings and have not been well described or optimized. To facilitate the study of follicular stem and progenitor cells and their interaction with surrounding skin, we have established a new murine transplantation model, similar to follicular unit transplantation in humans. Vibrissae from GFP transgenic mice were harvested, flip-side microdissected, and implanted individually into needle hole incisions in the back skin of immune-deficient nude mice. Grafts were evaluated histologically and the growth of transplanted vibrissae was observed. Transplanted follicles cycled spontaneously and newly formed hair shafts emerged from the skin after 2 weeks. Ninety percent of grafted vibrissae produced a hair shaft at 6 weeks. After pluck-induced follicle cycling, growth rates were equivalent to ungrafted vibrissae. Transplanted vibrissae with GFP-positive cells were easily identified in histological sections. We established a follicular vibrissa transplantation method that recapitulates human follicular unit transplantation. This method has several advantages over current protocols for animal hair transplantation. The method requires no suturing and minimizes the damage to donor follicles and recipient skin. Vibrissae are easier to microdissect and transplant than pelage follicles and, once transplanted, are readily distinguished from host pelage hair. This facilitates measurement of hair growth. Flip-side hair follicle microdissection precisely separates donor follicular tissue from interfollicular tissue and donor cells remain confined to hair follicles. This makes it possible to differentiate migration of hair follicle cells from interfollicular epidermis in lineage tracing wound experiments using genetically labeled donor follicles.

Macrophage phenotype in response to implanted synthetic scaffolds: an immunohistochemical study in the rat.

Macrophages predominate among the cells that directly interact with biomaterials and are key orchestrators of host-biomaterial interactions. However, the macrophage response to synthetic scaffolds in particular has not been well studied. The aim of this study was therefore to characterise the macrophage response to several synthetic scaffolds in the rat using immunohistological techniques for a panel of markers of macrophage subclass or activation, including ED1 (CD68), ED2 (CD163), CD80, mannose receptor and inducible nitric oxide synthase (iNOS). Materials were implanted subcutaneously and collected after 6-8 weeks during the chronic phase of the host response. Unmodified polycaprolactone scaffolds uniquely demonstrated a total lack of both macrophage adherence to surfaces and a wider foreign body response compared to scaffolds composed of poly(lactic-co-glycolic acid) (PLGA) and polyurethanes (PURs), with those macrophages present having a clear M2 (MR+, CD80-, iNOS-) phenotype. PLGA scaffolds displayed an M1-dominant (CD80+, iNOS+, MR-) response with substantial foreign body giant cell (FBGC) formation, whilst PUR scaffold FBGCs had a more mixed M1 (CD80+, iNOS+) and M2 (MR+) phenotype. The study also identified that the use of the ED1 antibody in the rat as a pan-macrophage marker is problematic as there is a separate and substantial ED2-positive macrophage population that it does not label, both in response to biomaterials and in normal tissues. The biomaterial-dependent nature of activation for both macrophages and FBGCs was confirmed, and nuanced M1/M2 phenotypes were described.

Liver specification of human iPSC-derived endothelial cells transplanted into mouse liver.

Background & Aims: Liver sinusoidal endothelial cells (LSECs) are important in liver development, regeneration, and pathophysiology, but the differentiation process underlying their tissue-specific phenotype is poorly understood and difficult to study because primary human cells are scarce. The aim of this study was to use human induced pluripotent stem cell (hiPSC)-derived LSEC-like cells to investigate the differentiation process of LSECs. Methods: hiPSC-derived endothelial cells (iECs) were transplanted into the livers of Fah-/-/Rag2-/-/Il2rg-/- mice and assessed over a 12-week period. Lineage tracing, immunofluorescence, flow cytometry, plasma human factor VIII measurement, and bulk and single cell transcriptomic analysis were used to assess the molecular and functional changes that occurred following transplantation. Results: Progressive and long-term repopulation of the liver vasculature occurred as iECs expanded along the sinusoids between hepatocytes and increasingly produced human factor VIII, indicating differentiation into LSEC-like cells. To chart the developmental profile associated with LSEC specification, the bulk transcriptomes of transplanted cells between 1 and 12 weeks after transplantation were compared against primary human adult LSECs. This demonstrated a chronological increase in LSEC markers, LSEC differentiation pathways, and zonation. Bulk transcriptome analysis suggested that the transcription factors NOTCH1, GATA4, and FOS have a central role in LSEC specification, interacting with a network of 27 transcription factors. Novel markers associated with this process included EMCN and CLEC14A. Additionally, single cell transcriptomic analysis demonstrated that transplanted iECs at 4 weeks contained zonal subpopulations with a region-specific phenotype. Conclusions: Collectively, this study confirms that hiPSCs can adopt LSEC-like features and provides insight into LSEC specification. This humanised xenograft system can be applied to further interrogate LSEC developmental biology and pathophysiology, bypassing current logistical obstacles associated with primary human LSECs. Impact and implications: Liver sinusoidal endothelial cells (LSECs) are important cells for liver biology, but better model systems are required to study them. We present a pluripotent stem cell xenografting model that produces human LSEC-like cells. A detailed and longitudinal transcriptomic analysis of the development of LSEC-like cells is included, which will guide future studies to interrogate LSEC biology and produce LSEC-like cells that could be used for regenerative medicine.

Engineering transplantable human lymphatic and blood capillary networks in a porous scaffold.

Due to a relative paucity of studies on human lymphatic assembly in vitro and subsequent in vivo transplantation, capillary formation and survival of primary human lymphatic (hLEC) and blood endothelial cells (hBEC) ± primary human vascular smooth muscle cells (hvSMC) were evaluated and compared in vitro and in vivo. hLEC ± hvSMC or hBEC ± hvSMC were seeded in a 3D porous scaffold in vitro, and capillary percent vascular volume (PVV) and vascular density (VD)/mm2 assessed. Scaffolds were also transplanted into a sub-cutaneous rat wound with morphology/morphometry assessment. Initially hBEC formed a larger vessel network in vitro than hLEC, with interconnected capillaries evident at 2 days. Interconnected lymphatic capillaries were slower (3 days) to assemble. hLEC capillaries demonstrated a significant overall increase in PVV (p = 0.0083) and VD (p = 0.0039) in vitro when co-cultured with hvSMC. A similar increase did not occur for hBEC + hvSMC in vitro, but hBEC + hvSMC in vivo significantly increased PVV (p = 0.0035) and VD (p = 0.0087). Morphology/morphometry established that hLEC vessels maintained distinct cell markers, and demonstrated significantly increased individual vessel and network size, and longer survival than hBEC capillaries in vivo, and established inosculation with rat lymphatics, with evidence of lymphatic function. The porous polyurethane scaffold provided advantages to capillary network formation due to its large (300-600 µm diameter) interconnected pores, and sufficient stability to ensure successful surgical transplantation in vivo. Given their successful survival and function in vivo within the porous scaffold, in vitro assembled hLEC networks using this method are potentially applicable to clinical scenarios requiring replacement of dysfunctional or absent lymphatic networks.

An "off the shelf" vascular allograft supports angiogenic growth in three-dimensional tissue engineering.

OBJECTIVES: Dense angiogenic sprouting occurs from arteriovenous loops (AVLs) incorporating autologous vein grafts inserted into empty plastic chambers in vivo. The purpose of this study was to determine if angiogenesis from the AVL was limited by substituting an "off the shelf" cold-stored allograft vein instead of an autologous vein. METHODS: Four Sprague Dawley rat groups (two AVL configurations × two chamber types) were established for both 2-week and 6-week harvest. Control AVLs were autologous femoral vein grafts harvested from the left femoral vein that were surgically inserted between the cut femoral artery and vein on the right side. Experimental "allograft" AVLs were rat femoral veins cold-stored (4°C, sterile) for 4 to 7 weeks and then microsurgically interposed between the right femoral artery and vein of an unrelated rat. The two AVL types were inserted in one of two plastic chamber types--smooth or perforated. At harvest, the AVL constructs were checked for patency, weighed, their volume determined, and histology undertaken. Morphometric assessment of percent and absolute volume of major tissue components (including blood vessels) at 6 weeks was completed. RESULTS: There were no significant differences between autograft and allograft groups in construct weight, volume, or morphology at 2 or 6 weeks. No statistical differences occurred in the percent or absolute vascular volume of AVLs incorporating a cold-stored allograft vs autologous vein grafts at 6 weeks regardless of the chamber type. However, perforated chambers caused significant increases in construct weight (P = .015), volume (P = .006), and percent and absolute connective tissue volume at 6 weeks (P = .001) compared to smooth chamber constructs, regardless of the graft type. CONCLUSION: Cold-stored small-caliber allografts interposed in AVLs do not inhibit microcirculatory development and can be used in composite tissue engineering.

Toward clinical application of stem cells for cardiac regeneration.

Heart failure affects more than 10% of the Australian population over age 65, and the ageing population will ensure continued growth of this significant problem. There are various treatment options available, but the growing field of regenerative therapy offers promise to restore or replace tissue lost in those with either congenital or acquired cardiac defects. Stem cells have many potential properties, but they need multiple discussed qualities to succeed in this field such as ease of harvest and multiplication, and most importantly minimal ethical concerns. There are multiple cell types available and one of the challenges will be to find the most appropriate cell type for cardiac regeneration. Cardiac tissue engineering is being explored using both in vitro and in vivo techniques. In vitro methods are primarily limited in terms of the vascularisation and size of the construct. In vivo engineered constructs overcome these limitations in early models, but they are still not ready for human trials. This review aims to provide the reader with an outline of the cell-based and tissue engineering therapies currently being used and developed for cardiac regeneration, as well as some insight into the potential problems that may hamper its progress in the future.

Differentiation of human adipose-derived stem cells into beating cardiomyocytes.

Human adipose-derived stem cells (ASCs) may differentiate into cardiomyocytes and this provides a source of donor cells for tissue engineering. In this study, we evaluated cardiomyogenic differentiation protocols using a DNA demethylating agent 5-azacytidine (5-aza), a modified cardiomyogenic medium (MCM), a histone deacetylase inhibitor trichostatin A (TSA) and co-culture with neonatal rat cardiomyocytes. 5-aza treatment reduced both cardiac actin and TropT mRNA expression. Incubation in MCM only slightly increased gene expression (1.5- to 1.9-fold) and the number of cells co-expressing nkx2.5/sarcomeric alpha-actin (27.2% versus 0.2% in control). TSA treatment increased cardiac actin mRNA expression 11-fold after 1 week, which could be sustained for 2 weeks by culturing cells in cardiomyocyte culture medium. TSA-treated cells also stained positively for cardiac myosin heavy chain, alpha-actin, TropI and connexin43; however, none of these treatments produced beating cells. ASCs in non-contact co-culture showed no cardiac differentiation; however, ASCs co-cultured in direct contact co-culture exhibited a time-dependent increase in cardiac actin mRNA expression (up to 33-fold) between days 3 and 14. Immunocytochemistry revealed co-expression of GATA4 and Nkx2.5, alpha-actin, TropI and cardiac myosin heavy chain in CM-DiI labelled ASCs. Most importantly, many of these cells showed spontaneous contractions accompanied by calcium transients in culture. Human ASC (hASC) showed synchronous Ca(2+) transient and contraction synchronous with surrounding rat cardiomyocytes (106 beats/min.). Gap junctions also formed between them as observed by dye transfer. In conclusion, cell-to-cell interaction was identified as a key inducer for cardiomyogenic differentiation of hASCs. This method was optimized by co-culture with contracting cardiomyocytes and provides a potential cardiac differentiation system to progress applications for cardiac cell therapy or tissue engineering.

Retrograde axonal tracing using manganese enhanced magnetic resonance imaging.

Manganese-enhanced magnetic resonance imaging (MEMRI) was used to investigate retrograde axonal tracing in the rat sciatic nerve model to assess its potential to examine peripheral nerve injury. The right sciatic nerve was exposed and crushed. After each recovery period, the distal part of the right sciatic nerve was injected with manganese (400 mM, 15 microl). After allowing 3 days for manganese transport the animals were subsequently scanned to visualize the sciatic nerve and its corresponding spinal cord and dorsal root ganglia with T1-weighted MRI. Thirty-four animals were randomly divided into 4 experimental groups according to their recovery period post-crush injury: 3 days (n=6), 2 weeks (n=6), 4 weeks (n=6) and 12 weeks (n=6); and two control groups: a non-crushed group (n=6) and a nerve cut group (n=4). In the no-injury group, the right sciatic nerve tract including its corresponding spinal cord and dorsal root ganglia showed significant T1 signal enhancement. In the animals with crush injury, the MR signal intensity was significantly reduced proximal to the injured site but gradually reappeared with increasing recovery period. The signal intensity of the sciatic tract was compared to the results of behavioral functional testing, retrograde axonal tracing with neural tracer fluorogold and histomorphometric analysis of the distal nerve. Significant correlations were observed between the MR signal intensity and the behavioral functional test (r=0.50, p<0.05), and the retrograde axonal tracing (r=0.88; p<0.05). Retrograde neuronal tract tracing with MEMRI can be used for the assessment of peripheral nerve damage and regeneration.

Disparate companions: tissue engineering meets cancer research.

Recreating an environment that supports and promotes fundamental homeostatic mechanisms is a significant challenge in tissue engineering. Optimizing cell survival, proliferation, differentiation, apoptosis and angiogenesis, and providing suitable stromal support and signalling cues are keys to successfully generating clinically useful tissues. Interestingly, those components are often subverted in the cancer setting, where aberrant angiogenesis, cellular proliferation, cell signalling and resistance to apoptosis drive malignant growth. In contrast to tissue engineering, identifying and inhibiting those pathways is a major challenge in cancer research. The recent discovery of adult tissue-specific stem cells has had a major impact on both tissue engineering and cancer research. The unique properties of these cells and their role in tissue and organ repair and regeneration hold great potential for engineering tissue-specific constructs. The emerging body of evidence implicating stem cells and progenitor cells as the source of oncogenic transformation prompts caution when using these cells for tissue-engineering purposes. While tissue engineering and cancer research may be considered as opposed fields of research with regard to their proclaimed goals, the compelling overlap in fundamental pathways underlying these processes suggests that cross-disciplinary research will benefit both fields. In this review article, tissue engineering and cancer research are brought together and explored with regard to discoveries that may be of mutual benefit.

Maxillofacial osseous reconstruction using the angular branch of the thoracodorsal vessels.

Mandibular and maxillary resections can produce complex three-dimensional defects requiring skeletal, soft tissue, and epithelial reconstruction. The subscapular vascular axis offers a source of skin, bone, and muscle on a single pedicle for microvascular flap transfer. We reviewed four cases where the subscapular vascular pedicle was used as a source of tissue for complex facial reconstructions in maxillofacial defects. Reconstruction of these complex defects was performed with a latissimus dorsi muscle or myocutaneous flap in combination with the lateral border of the scapula, harvested on the angular branch of the thoracodorsal vessels. There were three cases of maxillectomy and one case of partial mandibulectomy for malignant tumors. In each case, the angular branch of the thoracodorsal artery supplied 6 to 8 cm of the lateral border of the scapula and a latissimus dorsi myocutaneous flap was used for soft tissue reconstruction. Follow-up ranged from 9 months to 3 years and in all cases there was successful bony union. Shoulder movement was normal. This series encourages the further use of subscapular axis flaps as flexible sources of combined myocutaneous and osseous flaps on a single vascular pedicle in cases of complex maxillofacial reconstruction.

Liver sinusoidal endothelial cells promote the differentiation and survival of mouse vascularised hepatobiliary organoids.

The structural and physiological complexity of currently available liver organoids is limited, thereby reducing their relevance for drug studies, disease modelling, and regenerative therapy. In this study we combined mouse liver progenitor cells (LPCs) with mouse liver sinusoidal endothelial cells (LSECs) to generate hepatobiliary organoids with liver-specific vasculature. Organoids consisting of 5x103 cells were created from either LPCs, or a 1:1 combination of LPC/LSECs. LPC organoids demonstrated mild hepatobiliary differentiation in vitro with minimal morphological change; in contrast LPC/LSEC organoids developed clusters of polygonal hepatocyte-like cells and biliary ducts over a 7 day period. Hepatic (albumin, CPS1, CYP3A11) and biliary (GGT1) genes were significantly upregulated in LPC/LSEC organoids compared to LPC organoids over 7 days, as was albumin secretion. LPC/LSEC organoids also had significantly higher in vitro viability compared to LPC organoids. LPC and LPC/LSEC organoids were transplanted into vascularised chambers created in Fah-/-/Rag2-/-/Il2rg-/- mice (50 LPC organoids, containing 2.5x105 LPCs, and 100 LPC/LSEC organoids, containing 2.5x105 LPCs). At 2 weeks, minimal LPCs survived in chambers with LPC organoids, but robust hepatobiliary ductular tissue was present in LPC/LSEC organoids. Morphometric analysis demonstrated a 115-fold increase in HNF4α+ cells in LPC/LSEC organoid chambers (17.26 ± 4.34 cells/mm2 vs 0.15 ± 0.15 cells/mm2, p = 0.018), and 42-fold increase in Sox9+ cells in LPC/LSEC organoid chambers (28.29 ± 6.05 cells/mm2 vs 0.67 ± 0.67 cells/mm2, p = 0.011). This study presents a novel method to develop vascularised hepatobiliary organoids, with both in vitro and in vivo results confirming that incorporating LSECs with LPCs into organoids significantly increases the differentiation of hepatobiliary tissue within organoids and their survival post-transplantation.

Vascular Pedicle and Microchannels: Simple Methods Toward Effective In Vivo Vascularization of 3D Scaffolds.

Poor vascularization remains a key limiting factor in translating advances in tissue engineering to clinical applications. Vascular pedicles (large arteries and veins) isolated in plastic chambers are known to sprout an extensive capillary network. This study examined the effect vascular pedicles and scaffold architecture have on vascularization and tissue integration of implanted silk scaffolds. Porous silk scaffolds with or without microchannels are manufactured to support implantation of a central vascular pedicle, without a chamber, implanted in the groin of Sprague Dawley rats, and assessed morphologically and morphometrically at 2 and 6 weeks. At both time points, blood vessels, connective tissue, and an inflammatory response infiltrate all scaffold pores externally, and centrally when a vascular pedicle is implanted. At week 2, vascular pedicles significantly increase the degree of scaffold tissue infiltration, and both the pedicle and the scaffold microchannels significantly increase vascular volume and vascular density. Interestingly, microchannels contribute to increased scaffold vascularity without affecting overall tissue infiltration, suggesting a direct effect of biomaterial architecture on vascularization. The inclusion of pedicles and microchannels are simple and effective proangiogenic techniques for engineering thick tissue constructs as both increase the speed of construct vascularization in the early weeks post in vivo implantation.

Bio-engineering a tissue flap utilizing a porous scaffold incorporating a human induced pluripotent stem cell-derived endothelial cell capillary network connected to a vascular pedicle.

Tissue flaps are used to cover large/poorly healing wounds, but involve complex surgery and donor site morbidity. In this study a tissue flap is assembled using the mammalian body as a bioreactor to functionally connect an artery and vein to a human capillary network assembled from induced pluripotent stem cell-derived endothelial cells (hiPSC ECs). In vitro: Porous NovoSorb™ scaffolds (3 mm × 1.35 mm) were seeded with 200,000 hiPSC ECs ±â€¯100,000 human vascular smooth muscle cells (hvSMC), and cultured for 1-3 days, with capillaries formed by 24 h which were CD31+, VE-Cadherin+, EphB4+, VEGFR2+ and Ki67+, whilst hvSMCs (calponin+) attached abluminally. In vivo: In SCID mice, bi-lateral epigastric vascular pedicles were isolated in a silicone chamber for a 3 week 'delay period' for pedicle capillary sprouting, then reopened, and two hiPSC EC ±â€¯hvSMCs seeded scaffolds transplanted over the pedicle. The chamber was either resealed (Group 1), or removed and surrounding tissue secured around the pedicle + scaffolds (Group 2), for 1 or 2 weeks. Human capillaries survived in vivo and were CD31+, VE-Cadherin+ and VEGFR2+. Human vSMCs remained attached, and host mesenchymal cells also attached abluminally. Systemically injected FITC-dextran present in human capillary lumens indicated inosculation to host capillaries. Human iPSC EC capillary morphometric parameters at one week in vivo were equal to or higher than the same parameters measured in human abdominal skin. This 'proof of concept' study has demonstrated that bio-engineering an autologous human tissue flap based on hiPSC EC could minimize the use of donor flaps and has potential applications for complex wound coverage. STATEMENT OF SIGNIFICANCE: Tissue flaps, used for surgical reconstruction of wounds, require complex surgery, often associated with morbidity. Bio-engineering a simpler alternative, we assembled a human induced pluripotent stem cell derived endothelial cell (hiPSC ECs) capillary network in a porous scaffold in vitro, which when transplanted over a mouse vascular pedicle in vivo formed a functional tissue flap with mouse blood flow in the human capillaries. Therefore it is feasible to form an autologous tissue flap derived from a hiPSC EC capillary network assembled in vitro, and functionally connect to a vascular pedicle in vivo that could be utilized in complex wound repair for chronic or acute wounds.

Selenium nanoparticles as anti-infective implant coatings for trauma orthopedics against methicillin-resistant Staphylococcus aureus and epidermidis: in vitro and in vivo assessment.

Background: Bacterial infection is a common and serious complication in orthopedic implants following traumatic injury, which is often associated with extensive soft tissue damage and contaminated wounds. Multidrug-resistant bacteria have been found in these infected wounds, especially in patients who have multi trauma and prolonged stay in intensive care units.Purpose: The objective of this study was to develop a coating on orthopedic implants that is effective against drug-resistant bacteria. Methods and results: We applied nanoparticles (30-70nm) of the trace element selenium (Se) as a coating through surface-induced nucleation-deposition on titanium implants and investigated the antimicrobial activity against drug resistant bacteria including Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-resistant Staphylococcus epidermidis (MRSE) in vitro and in an infected femur model in rats.The nanoparticles were shown in vitro to have antimicrobial activity at concentrations as low as 0.5ppm. The nanoparticle coatings strongly inhibited biofilm formation on the implants and reduced the number of viable bacteria in the surrounding tissue following inoculation of implants with biofilm forming doses of bacteria. Conclusion: This study shows a proof of concept for a selenium nanoparticle coatings as a potential anti-infective barrier for orthopedic medical devices in the setting of contamination with multi-resistant bacteria. It also represents one of the few (if only) in vivo assessment of selenium nanoparticle coatings on reducing antibiotic-resistant orthopedic implant infections.

Cardiac tissue engineering in an in vivo vascularized chamber.

BACKGROUND: Cardiac tissue engineering offers the prospect of a novel treatment for acquired or congenital heart defects. We have created vascularized pieces of beating cardiac muscle in the rat that are as thick as the adult rat right ventricle wall. METHOD AND RESULTS: Neonatal rat cardiomyocytes in Matrigel were implanted with an arteriovenous blood vessel loop into a 0.5-mL patented tissue-engineering chamber, located subcutaneously in the groin. Chambers were harvested 1, 4, and 10 weeks after insertion. At 4 and 10 weeks, all constructs that grew in the chambers contracted spontaneously. Immunostaining for alpha-sarcomeric actin, troponin, and desmin showed that differentiated cardiomyocytes present in tissue at all time points formed a network of interconnected cells within a collagenous extracellular matrix. Constructs at 4 and 10 weeks were extensively vascularized. The maximum thickness of cardiac tissue generated was 1983 microm. Cardiomyocytes increased in size from 1 to 10 weeks and were positive for the proliferation markers Ki67 and PCNA. Connexin-43 stain indicated that gap junctions were present between cardiomyocytes at 4 and 10 weeks. Echocardiograms performed between 4 and 10 weeks showed that the tissue construct contracted spontaneously in vivo. In vitro organ bath experiments showed a typical cardiac muscle length-tension relationship, the ability to be paced from electrical field pulses up to 3 Hz, positive chronotropy to norepinephrine, and positive inotropy in response to calcium. CONCLUSIONS: In summary, the use of a vascularized tissue-engineering chamber allowed generation of a spontaneously beating 3-dimensional mass of cardiac tissue from neonatal rat cardiomyocytes. Further development of this vascularized model will increase the potential of cardiac tissue engineering to provide suitable replacement tissues for acquired and congenital defects.

An endogenously deposited fibrin scaffold determines construct size in the surgically created arteriovenous loop chamber model of tissue engineering.

BACKGROUND: An arteriovenous loop (AVL) enclosed in a polycarbonate chamber in vivo, produces a fibrin exudate which acts as a provisional matrix for the development of a tissue engineered microcirculatory network. OBJECTIVES: By administering enoxaparin sodium - an inhibitor of fibrin polymerization, the significance of fibrin scaffold formation on AVL construct size (including the AVL, fibrin scaffold, and new tissue growth into the fibrin), growth, and vascularization were assessed and compared to controls. METHODS: In Sprague Dawley rats, an AVL was created on femoral vessels and inserted into a polycarbonate chamber in the groin in 3 control groups (Series I) and 3 experimental groups (Series II). Two hours before surgery and 6 hours post-surgery, saline (Series I) or enoxaparin sodium (0.6 mg/kg, Series II) was administered intra-peritoneally. Thereafter, the rats were injected daily with saline (Series I) or enoxaparin sodium (1.5 mg/kg, Series II) until construct retrieval at 3, 10, or 21 days. The retrieved constructs underwent weight and volume measurements, and morphologic/morphometric analysis of new tissue components. RESULTS: Enoxaparin sodium treatment resulted in the development of smaller AVL constructs at 3, 10, and 21 days. Construct weight and volume were significantly reduced at 10 days (control weight 0.337 +/- 0.016 g [Mean +/- SEM] vs treated 0.228 +/- 0.048, [P < .001]: control volume 0.317 +/- 0.015 mL vs treated 0.184 +/- 0.039 mL [P < .01]) and 21 days (control weight 0.306 +/- 0.053 g vs treated 0.198 +/- 0.043 g [P < .01]: control volume 0.285 +/- 0.047 mL vs treated 0.148 +/- 0.041 mL, [P < .01]). Angiogenesis was delayed in the enoxaparin sodium-treated constructs with the absolute vascular volume significantly decreased at 10 days (control vascular volume 0.029 +/- 0.03 mL vs treated 0.012 +/- 0.002 mL [P < .05]). CONCLUSION: In this in vivo tissue engineering model, endogenous, extra-vascularly deposited fibrin volume determines construct size and vascular growth in the first 3 weeks and is, therefore, critical to full construct development.

Adipose differentiation of bone marrow-derived mesenchymal stem cells using Pluronic F-127 hydrogel in vitro.

Due to increasing clinical demand for adipose tissue, a suitable scaffold for engineering adipose tissue constructs is needed. In this study, we have developed a three-dimensional (3-D) culture system using bone marrow-derived mesenchymal stem cells (BM-MSC) and a Pluronic F-127 hydrogel scaffold as a step towards the in vitro tissue engineering of fat. BM-MSC were dispersed into a Pluronic F-127 hydrogel with or without type I collagen added. The adipogenic differentiation of the BM-MSC was assessed by cellular morphology and further confirmed by Oil Red O staining. The BM-MSC differentiated into adipocytes in Pluronic F-127 in the presence of adipogenic stimuli over a period of 2 weeks, with some differentiation present even in absence of such stimuli. The addition of type I collagen to the Pluronic F-127 caused the BM-MSC to aggregate into clumps, thereby generating an uneven adipogenic response, which was not desirable.

An arteriovenous loop in a protected space generates a permanent, highly vascular, tissue-engineered construct.

A major obstacle to 3-dimensional tissue engineering is incorporation of a functional vascular supply to support the expanding new tissue. This is overcome in an in vivo intrinsic vascularization model where an arteriovenous loop (AVL) is placed in a noncollapsible space protected by a polycarbonate chamber. Vascular development and hypoxia were examined from 3 days to 112 days by vascular casting, morphometric, and morphological techniques to understand the model's vascular growth and remodeling parameters for tissue engineering purposes. At 3 days a fibrin exudate surrounded the AVL, providing a scaffold to migrating inflammatory, endothelial, and mesenchymal cells. Capillaries formed between 3 and 7 days. Hypoxia and cell proliferation were maximal at 7 days, followed by a peak in percent vascular volume at 10 days (23.20+/-3.14% compared with 3.59+/-2.68% at 3 days, P<0.001). Maximal apoptosis was observed at 112 days. The protected space and spontaneous microcirculatory development in this model suggest it would be applicable for in vivo tissue engineering. A temporal window in a period of intense angiogenesis at 7 to 10 days is optimal for exogenous cell seeding and survival in the chamber, potentially enabling specific tissue outcomes to be achieved.