Peroxide-based oxygen generating topical wound dressing for enhancing healing of dermal wounds.

Oxygen generating biomaterials represent a new trend in regenerative medicine that aims to generate and supply oxygen at the site of requirement, to support tissue healing and regeneration. To enhance the healing of dermal wounds, we have developed a highly portable, in situ oxygen generating wound dressings that uses sodium percarbonate (SPO) and calcium peroxide (CPO) as chemical oxygen sources. The dressing continuously generated oxygen for more than 3 days, after which it was replaced. In the in vivo testing on porcine full-thickness porcine wound model, the SPO/CPO dressing showed enhanced wound healing during the 8 week study period. Quantitative measurements of wound healing related parameters, such as wound closure, reepithelialization, epidermal thickness and collagen content of dermis showed that supplying oxygen topically using the SPO/CPO dressing significantly accelerated the wound healing. An increase in neovascularization, as determined using Von Willebrand factor (vWF) and CD31 staining, was also observed in the presence of SPO/CPO dressing. This novel design for a wound dressing that contains oxygen generating biomaterials (SPO/CPO) for supplying topical oxygen, may find utility in treating various types of acute to chronic wounds.

Combined systemic and local delivery of stem cell inducing/recruiting factors for in situ tissue regeneration.

Whereas the conventional tissue engineering strategy involves the use of scaffolds combined with appropriate cell types to restore normal functions, the concept of in situ tissue regeneration uses host responses to a target-specific scaffold to mobilize host cells to a site of injury without the need for cell seeding. For this purpose, local delivery of bioactive molecules from scaffolds has been generally used. However, this approach has limited stem cell recruitment into the implants. Thus, we developed a combination of systemic delivery of substance P (SP) and local release of stromal-derived factor-1α (SDF-1α) from an implant. In this study, we examined whether this combined system would significantly enhance recruitment of host stem cells into the implants. Flow cytometry and immunohistochemistry for CD29/CD45, CD146/α-smooth muscle actin, and c-kit demonstrated that this system significantly increased the number of stem cell-like cells within the implants when compared with other systems. In vitro culture of the cells that had infiltrated into the scaffolds from the combined system confirmed that host stem cells were recruited into these implants and indicated that they were capable of differentiation into multiple lineages. These results indicate that this combined system may lead to more efficient tissue regeneration.

Regenerative medicine as applied to general surgery.

The present review illustrates the state of the art of regenerative medicine (RM) as applied to surgical diseases and demonstrates that this field has the potential to address some of the unmet needs in surgery. RM is a multidisciplinary field whose purpose is to regenerate in vivo or ex vivo human cells, tissues, or organs to restore or establish normal function through exploitation of the potential to regenerate, which is intrinsic to human cells, tissues, and organs. RM uses cells and/or specially designed biomaterials to reach its goals and RM-based therapies are already in use in several clinical trials in most fields of surgery. The main challenges for investigators are threefold: Creation of an appropriate microenvironment ex vivo that is able to sustain cell physiology and function in order to generate the desired cells or body parts; identification and appropriate manipulation of cells that have the potential to generate parenchymal, stromal and vascular components on demand, both in vivo and ex vivo; and production of smart materials that are able to drive cell fate.

Tissue-engineered autologous urethras for patients who need reconstruction: an observational study.

BACKGROUND: Complex urethral problems can occur as a result of injury, disease, or congenital defects and treatment options are often limited. Urethras, similar to other long tubularised tissues, can stricture after reconstruction. We aimed to assess the effectiveness of tissue-engineered urethras using patients' own cells in patients who needed urethral reconstruction. METHODS: Five boys who had urethral defects were included in the study. A tissue biopsy was taken from each patient, and the muscle and epithelial cells were expanded and seeded onto tubularised polyglycolic acid:poly(lactide-co-glycolide acid) scaffolds. Patients then underwent urethral reconstruction with the tissue-engineered tubularised urethras. We took patient history, asked patients to complete questionnaires from the International Continence Society (ICS), and did urine analyses, cystourethroscopy, cystourethrography, and flow measurements at 3, 6, 12, 24, 36, 48, 60, and 72 months after surgery. We did serial endoscopic cup biopsies at 3, 12, and 36 months, each time in a different area of the engineered urethras. FINDINGS: Patients had surgery between March 19, 2004, and July 20, 2007. Follow-up was completed by July 31, 2010. Median age was 11 years (range 10-14) at time of surgery and median follow-up was 71 months (range 36-76 months). AE1/AE3, α actin, desmin, and myosin antibodies confirmed the presence of cells of epithelial and muscle lineages on all cultures. The median end maximum urinary flow rate was 27·1 mL/s (range 16-28), and serial radiographic and endoscopic studies showed the maintenance of wide urethral calibres without strictures. Urethral biopsies showed that the engineered grafts had developed a normal appearing architecture by 3 months after implantation. INTERPRETATION: Tubularised urethras can be engineered and remain functional in a clinical setting for up to 6 years. These engineered urethras can be used in patients who need complex urethral reconstruction. FUNDING: National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health.

One and four layer acellular bladder matrix for fascial tissue reconstruction.

To determine whether the use of multiple layers of acellular bladder matrix (ABM) is more suitable for the treatment of abdominal wall hernia than a single layered ABM. The feasibility, biocompatibility and mechanical properties of both materials were assessed and compared. Biocompatibility testing was performed on 4 and 1 layered ABM. The matrices were used to repair an abdominal hernia model in 24 rabbits. The animals were followed for up to 3 months. Immediately after euthanasia, the implant site was inspected and samples were retrieved for histology, scanning electron microscopy and biomechanical studies. Both acellular biomaterials demonstrated excellent biocompatibility. At the time of retrieval, there was no evidence of infection. The matrices demonstrated biomechanical properties comparable to native tissue. Three hernias (25%) were found in the single layer ABM group and only 1 hernia (8%) was found in the 4 layer ABM group. Histologically, the matrix structure was intact and the cell density within the matrices decreased with time. The dominant cell type present within the matrices shifted from lymphocytes to fibroblasts over time. Both ABMs maintained adequate strength over time when used for hernia repair, and there was an extremely low incidence of adhesion formation. The single layer ABM showed enhanced cellular integration, while the 4 layer ABM reduced hernia formation. Either of these matrices may be useful as an off-the-shelf biomaterial for patients requiring fascial repair.

Self-Assembling Peptide Solution Accelerates Hemostasis.

Objective: One of the leading causes of death following traumatic injury is exsanguination. Biological material-based hemostatic agents such as fibrin, thrombin, and albumin have a high risk for causing infection. Synthetic peptide-based hemostatic agents offer an attractive alternative. The objective of this study is to explore the potential of h9e peptide as an effective hemostatic agent in both in vitro and in vivo models. Approach:In vitro blood coagulation kinetics in the presence of h9e peptide was determined as a function of gelation time using a dynamic rheometer. In vivo hemostatic effects were studied using the Wistar rat model. Results were compared to those of the commercial hemostatic product Celox™, a chitosan-based product. Adhesion of h9e peptide was evaluated using the platelet adhesion test. Biocompatibility of h9e peptide was studied in vivo using a mouse model. Results: After h9e peptide solution was mixed with blood, gelation started immediately, increased rapidly with time, and reached more than 100 Pa within 3 s. Blood coagulation strength increased as h9e peptide wt% concentration increased. In the rat model, h9e peptide solution at 5% weight concentration significantly reduced both bleeding time and blood loss, outperforming Celox. Preliminary pathological studies indicate that h9e peptide solution is biocompatible and did not have negative effects when injected subcutaneously in a mouse model. Innovation: For the first time, h9e peptide was found to have highly efficient hemostatic effects by forming nanoweb-like structures, which act as a preliminary thrombus and a surface to arrest bleeding 82% faster compared to the commercial hemostatic agent Celox. Conclusion: This study demonstrates that h9e peptide is a promising hemostatic biomaterial, not only because of its greater hemostatic effect than commercial product Celox but also because of its excellent biocompatibility based on the in vivo mouse model study.

Hedgehog signaling is required at multiple stages of zebrafish tooth development.

BACKGROUND: The accessibility of the developing zebrafish pharyngeal dentition makes it an advantageous system in which to study many aspects of tooth development from early initiation to late morphogenesis. In mammals, hedgehog signaling is known to be essential for multiple stages of odontogenesis; however, potential roles for the pathway during initiation of tooth development or in later morphogenesis are incompletely understood. RESULTS: We have identified mRNA expression of the hedgehog ligands shha and the receptors ptc1 and ptc2 during zebrafish pharyngeal tooth development. We looked for, but did not detect, tooth germ expression of the other known zebrafish hedgehog ligands shhb, dhh, ihha, or ihhb, suggesting that as in mammals, only Shh participates in zebrafish tooth development. Supporting this idea, we found that morphological and gene expression evidence of tooth initiation is eliminated in shha mutant embryos, and that morpholino antisense oligonucleotide knockdown of shha, but not shhb, function prevents mature tooth formation. Hedgehog pathway inhibition with the antagonist compound cyclopamine affected tooth formation at each stage in which we applied it: arresting development at early stages and disrupting mature tooth morphology when applied later. These results suggest that hedgehog signaling is required continuously during odontogenesis. In contrast, over-expression of shha had no effect on the developing dentition, possibly because shha is normally extensively expressed in the zebrafish pharyngeal region. CONCLUSION: We have identified previously unknown requirements for hedgehog signaling for early tooth initiation and later morphogenesis. The similarity of our results with data from mouse and other vertebrates suggests that despite gene duplication and changes in the location of where teeth form, the roles of hedgehog signaling in tooth development have been largely conserved during evolution.

Composite scaffolds for the engineering of hollow organs and tissues.

Several types of synthetic and naturally derived biomaterials have been used for augmenting hollow organs and tissues. However, each has desirable traits which were exclusive of the other. We fabricated a composite scaffold and tested its potential for the engineering of hollow organs in a bladder tissue model. The composite scaffolds were configured to accommodate a large number of cells on one side and were designed to serve as a barrier on the opposite side. The scaffolds were fabricated by bonding a collagen matrix to PGA polymers with threaded collagen fiber stitches. Urothelial and bladder smooth muscle cells were seeded on the composite scaffolds, and implanted in mice for up to 4 weeks and analyzed. Both cell types readily attached and proliferated on the scaffolds and formed bladder tissue-like structures in vivo. These structures consisted of a luminal urothelial layer, a collagen rich compartment and a peripheral smooth muscle layer. Biomechanical studies demonstrated that the tissues were readily elastic while maintaining their pre-configured structures. This study demonstrates that a composite scaffold can be fabricated with two completely different polymer systems for the engineering of hollow organs. The composite scaffolds are biocompatible, possess adequate physical and structural characteristics for bladder tissue engineering, and are able to form tissues in vivo. This scaffold system may be useful in patients requiring hollow organ replacement.

The effect of BMP-mimetic peptide tethering bioinks on the differentiation of dental pulp stem cells (DPSCs) in 3D bioprinted dental constructs.

The goal of this study was to use 3D bioprinting technology to create a bioengineered dental construct containing human dental pulp stem cells (hDPSCs). To accomplish this, we first developed a novel bone morphogenetic protein (BMP) peptide-tethering bioink formulation and examined its rheological properties, its printability, and the structural stability of the bioprinted construct. Second, we evaluated the survival and differentiation of hDPSCs in the bioprinted dental construct by measuring cell viability, proliferation, and gene expression, as well as histological and immunofluorescent analyses. Our results showed that the peptide conjugation into the gelatin methacrylate-based bioink formulation was successfully performed. We determined that greater than 50% of the peptides remained in the bioprinted construct after three weeks in vitro cell culture. Human DPSC viability was >90% in the bioprinted constructs immediately after the printing process. Alizarin Red staining showed that the BMP peptide construct group exhibited the highest calcification as compared to the growth medium, osteogenic medium, and non-BMP peptide construct groups. In addition, immunofluorescent and quantitative reverse transcription-polymerase chain reaction analyses showed robust expression of dentin sialophosphoprotein and osteocalcin in the BMP peptide dental constructs. Together, these results strongly suggested that BMP peptide-tethering bioink could accelerate the differentiation of hDPSCs in 3D bioprinted dental constructs.

Effect of Hierarchical Scaffold Consisting of Aligned dECM Nanofibers and Poly(lactide-co-glycolide) Struts on the Orientation and Maturation of Human Muscle Progenitor Cells.

Extracellular matrices (ECMs) derived from tissues and decellularized are widely used as biomaterials in tissue engineering applications because they encompass tissue-specific biological and physical cues. In this study, we utilized a solubilized decellularized tissue (dECM) obtained from skeletal muscle to fabricate a nanofibrous structure using the electrospinning technique. The dECM was chemically modified by methacrylate reaction (dECM-MA) to improve the structural stability before electrospinning. The electrospun dECM-MA nanofibers were combined with microscale fibrillated poly(lactide-co-glycolide) (PLGA) constructs fabricated by three-dimensional printing and fibrillation/leaching of poly(vinyl alcohol) to promote skeletal muscle cell orientation and maturation. Using the electrostatic force-assisted fiber-alignment method, a multiscale composite scaffold consisting of fibrillated PLGA and aligned dECM-MA nanofibers was fabricated. The multiscale dECM-MA/PLGA composite scaffold significantly promoted the cellular orientation and myotube formation of human muscle progenitor cells compared to control scaffolds. The results suggested the potential use of the multiscale dECM-MA/PLGA composite scaffold, which contains the biochemical and topographical cues, for bioengineering a skeletal muscle tissue construct.

Tissue engineering a complete vaginal replacement from a small biopsy of autologous tissue.

BACKGROUND: In women, a healthy, patent vagina is important for the maintenance of a good quality of life. Apart from congenital abnormalities, such as cloacal exstrophy, intersex disorders, and an absence of the posterior two thirds of the organ, individuals may also suffer from cancer, trauma, infection, inflammation, or iatrogenic injuries leading to tissue damage and loss -- all of which require vaginal repair or replacement. Of necessity, reconstruction is often performed with nonvaginal tissue substitutes, such as segments of large intestine or skin, which are not anatomically or functionally ideal (Hendren and Atala, J Urol 1994; 152: 752; Hendren and Atala, J Pediatr Surg 1995; 30: 91). Whenever such tissue is used additional complications often ensue, such as strictures, infection, hair growth, graft shrinkage, diverticuli, and even malignancy (Filipas et al., BJU Int 2000; 85: 715; Lai and Chang, Changgeng Yi Xue Za Zhi 1999; 22: 253; Parsons et al., J Pediatr Surg 2002; 37: 629; Seccia et al., Ann Plast Surg 2002; 49: 379; Filipas, Curr Opin Urol 2001; 11: 267). METHODS: Using a rabbit model, we report here the construction of a functional vagina using autologous cells expanded from a small vaginal biopsy. RESULTS.: Six months after total vaginal replacement, radiographic analysis of rabbits implanted with the neovagina demonstrated wide, patent vaginal calibers without strictures. Histologic analysis revealed well-organized epithelial and muscle cell layers. Physiologic studies showed normal-range responses to electrical stimulation or to an adrenergic agonist. CONCLUSIONS: These data indicate that a tissue engineering approach to clinical vaginal reconstruction in women is now a realistic possibility.

Local and systemic effects of a tissue engineered neobladder in a canine cystoplasty model.

PURPOSE: Tissue engineered bladders are emerging as a potential treatment option in urological surgery. Although successful neobladders can be engineered with autologous cells on a biodegradable polymer scaffold, studies of the local and systemic effects on host tissue have not been extensively pursued. We examined such effects at predetermined time points after implantation of tissue engineered neobladders in a canine cystoplasty model. MATERIALS AND METHODS: Eight dogs underwent trigone sparing cystectomies. Six dogs (experimental group) received bladder augmentation with tissue engineered constructs produced from autologous urothelial and smooth muscle cells on a prefabricated polyglycolic acid polymer scaffold. Two beagles (control group) received bladder augmentation with the polyglycolic acid scaffold alone. Serial urodynamic studies, cystograms, peripheral blood smears, urinalysis, serum chemistry, complete blood count and electrolytes were done at predetermined time points postoperatively. The bladder, and local and distant organs were retrieved 6 months after surgery for analysis. RESULTS: Capacity and compliance of the engineered bladders reached normal levels by 6 months. Engineered bladders showed tissue composition similar to that of normal bladders. Infiltration of inflammatory cells was minimal and subsided with time. An increase in the total systemic leukocyte count and in bacteriuria was evident initially at 1 week but they returned to normal levels by 1 month postoperatively. Other systemic parameters remained within normal levels at all time points. There was no evidence of abnormal findings in local or distant organs. CONCLUSIONS: Implantation of polymer molds seeded with autologous bladder cells did not show significant local or systemic toxicity in a canine model. This study suggests that such engineered neobladders are safe and effective for reconstructive surgery.

The use of thermal treatments to enhance the mechanical properties of electrospun poly(epsilon-caprolactone) scaffolds.

Nonwoven nanofiber scaffolds fabricated by electrospinning technology have been widely used for tissue engineering applications. Although electrospun nanofiber scaffolds fulfill many requirements for tissue engineering applications, they sometimes lack the necessary biomechanical properties. To attempt to improve the biomechanical properties of electrospun poly(epsilon-caprolactone) (PCL) scaffolds, fibers were bonded by thermal treatment. The thermal fiber bonding was performed in Pluronic F127 solution at a range of temperatures from 54 degrees C to 60 degrees C. Thermally bonded electrospun PCL scaffolds were characterized by analyzing the changes in morphology, fiber diameter, pore area, tensile properties, suture retention strength, burst pressure strength, and compliance. The biomechanical properties of the thermally bonded electrospun PCL scaffolds were significantly increased without any gross observable and ultrastructural changes when compared to untreated PCL scaffolds. This study suggests that the introduction of thermal fiber bonding to electrospun PCL scaffolds improved the biomechanical properties of these scaffolds, making them more suitable for tissue engineering applications.

Development of a composite vascular scaffolding system that withstands physiological vascular conditions.

Numerous scaffolds that possess ideal characteristics for vascular grafts have been fabricated for clinical use. However, many of these scaffolds may not show consistent properties when they are exposed to physiologic vascular environments that include high pressure and flow, and they may eventually fail due to unexpected rapid degradation and low resistance to shear stress. There is a demand to develop a more durable scaffold that could withstand these conditions until vascular tissue matures in vivo. In this study, vascular scaffolds composed of poly(epsilon-caprolactone) (PCL) and collagen were fabricated by electrospinning. Morphological, biomechanical, and biological properties of these composite scaffolds were examined. The PCL/collagen composite scaffolds, with fiber diameters of approximately 520 nm, possessed appropriate tensile strength (4.0+/-0.4 MPa) and adequate elasticity (2.7+/-1.2 MPa). The burst pressure of the composite scaffolds was 4912+/-155 mmHg, which is much greater than that of the PCL-only scaffolds (914+/-130 mmHg) and native vessels. The composite scaffolds seeded with bovine endothelial cells (bECs) and smooth muscle cells (bSMCs) showed the formation of a confluent layer of bECs on the lumen and bSMCs on the outer surface of the scaffold. The PCL/collagen composite scaffolds are biocompatible, possess biomechanical properties that resist high degrees of pressurized flow over long term, and provide a favorable environment that supports the growth of vascular cells.

The influence of electrospun aligned poly(epsilon-caprolactone)/collagen nanofiber meshes on the formation of self-aligned skeletal muscle myotubes.

Current treatment options for restoring large skeletal muscle tissue defects due to trauma or tumor ablation are limited by the host muscle tissue availability and donor site morbidity of muscle flap implantation. Creation of implantable functional muscle tissue that could restore muscle defects may bea possible solution. To engineer functional muscle tissue for reconstruction, scaffolds that mimic native fibers need to be developed. In this study we examined the feasibility of using poly(epsilon-caprolactone) (PCL)/collagen based nanofibers using electrospinning as a scaffold system for implantable engineered muscle. We investigated whether electrospun nanofibers could guide morphogenesis of skeletal muscle cells and enhance cellular organization. Nanofibers with different fiber orientations were fabricated by electrospinning with a blend of PCL and collagen. Human skeletal muscle cells (hSkMCs) were seeded onto the electrospun PCL/collagen nanofiber meshes and analyzed for cell adhesion, proliferation and organization. Our results show that unidirectionally oriented nanofibers significantly induced muscle cell alignment and myotube formation as compared to randomly oriented nanofibers. The aligned composite nanofiber scaffolds seeded with skeletal muscle cells may provide implantable functional muscle tissues for patients with large muscle defects.

In Situ Tissue Regeneration of Renal Tissue Induced by Collagen Hydrogel Injection.

Host stem/progenitor cells can be mobilized and recruited to a target location using biomaterials, and these cells may be used for in situ tissue regeneration. The objective of this study was to investigate whether host biologic resources could be used to regenerate renal tissue in situ. Collagen hydrogel was injected into the kidneys of normal mice, and rat kidneys that had sustained ischemia/reperfusion injury. After injection, the kidneys of both animal models were examined up to 4 weeks for host tissue response. The infiltrating host cells present within the injection regions expressed renal stem/progenitor cell markers, PAX-2, CD24, and CD133, as well as mesenchymal stem cell marker, CD44. The regenerated renal structures were identified by immunohistochemistry for renal cell specific markers, including synaptopodin and CD31 for glomeruli and cytokeratin and neprilysin for tubules. Quantitatively, the number of glomeruli found in the injected regions was significantly higher when compared to normal regions of renal cortex. This phenomenon occurred in normal and ischemic injured kidneys. Furthermore, the renal function after ischemia/reperfusion injury was recovered after collagen hydrogel injection. These results demonstrate that introduction of biomaterials into the kidney is able to facilitate the regeneration of glomerular and tubular structures in normal and injured kidneys. Such an approach has the potential to become a simple and effective treatment for patients with renal failure. Stem Cells Translational Medicine 2018;7:241-250.

Biofabrication: A Guide to Technology and Terminology.

Biofabrication holds the potential to generate constructs that more closely recapitulate the complexity and heterogeneity of tissues and organs than do currently available regenerative medicine therapies. Such constructs can be applied for tissue regeneration or as in vitro 3D models. Biofabrication is maturing and growing, and scientists with different backgrounds are joining this field, underscoring the need for unity regarding the use of terminology. We therefore believe that there is a compelling need to clarify the relationship between the different concepts, technologies, and descriptions of biofabrication that are often used interchangeably or inconsistently in the current literature. Our objective is to provide a guide to the terminology for different technologies in the field which may serve as a reference for the biofabrication community.

Oxygen producing biomaterials for tissue regeneration.

A limiting factor in regenerating large organs and healing large wounds completely is the inability to provide oxygen to the affected areas for vascularization and healing to occur. An oxygen rich compound of sodium percarbonate was incorporated into films of Poly(D,L-lactide-co-glycolide) (PLGA) and used for in situ production of oxygen. Oxygen release could be observed from the film over a period of 24 h. When the oxygen producing biomaterials were placed in contact with ischemic tissue in a mouse model, decreased tissue necrosis and cellular apoptosis was observed. This indicates that improved tissue viability could be maintained for several days using oxygen producing biomaterials.

In vitro evaluation of electrospun nanofiber scaffolds for vascular graft application.

Blood vessels are diverse in size, mechanical and biochemical properties, cellular content, and ultrastructural organization depending on their location and specific function. Therefore, it is required to control the fabrication of vascular grafts for obtaining desirable characteristics of blood vessel substitutes. In this study we have fabricated various scaffolds using the electrospinning technique with blends of collagen, elastin, and several biodegradable polymers. Biocompatibility, dimensional stability in vitro and mechanical properties were evaluated. Materials were blended at a relative concentration by weight of 45% collagen, 15% elastin, and 40% synthetic polymer to mimic the ratio of collagen and elastin in native blood vessels. The fabricated scaffolds are composed of randomly oriented fibers with diameters ranging from 477 to 765 nm. The electrospun scaffolds are nontoxic, dimensionally stable in an in vitro culture environment, easily fabricated, and possess controlled mechanical properties that simulate the ultrastructure of native blood vessels. The present study suggests that the introduction of synthetic biodegradable polymers enabled tailoring of mechanical properties of vascular substitutes and improving compliance matching for vascular tissue engineering.

Generation of histocompatible tissues using nuclear transplantation.

Nuclear transplantation (therapeutic cloning) could theoretically provide a limitless source of cells for regenerative therapy. Although the cloned cells would carry the nuclear genome of the patient, the presence of mitochondria inherited from the recipient oocyte raises questions about the histocompatibility of the resulting cells. In this study, we created bioengineered tissues from cardiac, skeletal muscle, and renal cells cloned from adult bovine fibroblasts. Long-term viability was demonstrated after transplantation of the grafts into the nuclear donor animals. Reverse transcription-PCR (RT-PCR) and western blot analysis confirmed that the cloned tissues expressed tissue-specific mRNA and proteins while expressing a different mitochondrial DNA (mtDNA) haplotype. In addition to creating skeletal muscle and cardiac "patches", nuclear transplantation was used to generate functioning renal units that produced urinelike fluid and demonstrated unidirectional secretion and concentration of urea nitrogen and creatinine. Examination of the explanted renal devices revealed formation of organized glomeruli- and tubule-like structures. Delayed-type hypersensitivity (DTH) testing in vivo and Elispot analysis in vitro suggested that there was no rejection response to the cloned renal cells. The ability to generate histocompatible cells using cloning techniques addresses one of the major challenges in transplantation medicine.