Martin Meuli

Spontaneous repair of superficial defects in articular cartilage in a fetal lamb model

A fetal lamb model was developed to investigate the capacity of fetal articular cartilage for repair after the creation of a superficial defect. Superficial defects, 100 micrometers deep, were made in the articular cartilage of the trochlear groove in the distal aspect of the femur in eighteen fetal lambs that were halfway through the 145-day gestational period; the contralateral limb was used as a sham control. The wounds were allowed to heal in utero for three, seven, fourteen, twenty-one, or twenty-eight days. Seven days after the injury, the defects were filled with a hypocellular matrix, which stained lightly with safranin O. At twenty-eight days, the staining of the matrix was similar to that of the sham controls and the chondrocyte density and the architectural arrangement of the cell layers had been restored. An inflammatory response was not elicited, and no fibrous scar tissue was observed. CLINICAL RELEVANCE: An orderly sequence of repair of articular cartilage was observed after the creation of partial-thickness defects in the distal aspect of the femur of mid-gestational fetal lambs. The fetal lamb model may be useful for the investigation of interactions between the chondrocyte and extracellular matrices after mechanical stimulation. Fundamental knowledge of the metabolism of fetal articular cartilage may provide insight into latent reparative processes of mature cartilage.

The spinal cord lesion in human fetuses with myelomeningocele: Implications for fetal surgery

Recently produced experimental evidence suggests that secondary traumatic injury and degenerative changes, acquired in utero, to the openly exposed neural tissue may be primarily responsible for the massive neurological deficit associated with myelomeningocele (MMC). The goal of this study was to examine the morphology of human fetuses with MMC to determine if acquired trauma to the spinal cord could be identified. The MMC lesions with surrounding tissues from 10 human fetuses ranging in gestational age between 19 and 23 weeks were prepared with serial histological sections. The MMC lesions were characterized by an open vertebral arch, an open dura mater fused laterally to the dermis, and an open pia mater fused laterally to the epidermis. The spinal cord was exposed, without any meningeal, bony, or cutaneous covering, and was resting on the dorsal aspect of the abnormal arachnoid sac created by the fusion of the meninges to the cutaneous tissues. The exposed neural tissue had undergone varying degrees of recent traumatic injury as a result of its exposed position, ranging from nearly complete preservation of neural elements in four cases to nearly complete loss in two cases. The neural tissue remaining in the MMC with partial loss contained hemorrhages and abrasions from recent injury, suggesting that injury occurred during passage through the birth canal. The presence of dorsal and ventral parts of the cord with nerve roots and ganglia demonstrated that these structures had formed during development and that the loss of tissue by injury was a secondary change. The results support the concept that performing in utero surgery could protect the exposed but initially well-developed and uninjured cord, prevent secondary neural injury, and preserve neural function in the human fetus with myelomeningocele.

A model of scarless human fetal wound repair is deficient in transforming growth factor beta

Human fetal skin heals via scarless regeneration, whereas adult skin heals with scar. Scarless repair may reflect a distinct cytokine milieu. We studied the role of the cytokine transforming growth factor beta (TGF beta) using an established model of scarless human fetal skin repair in which human fetal skin is transplanted into a subcutaneous pocket on the flank of an adult nude mouse. In this model, wounded 16-week-gestation human fetal skin heals without scar, whereas wounded adult skin heals with scar. Seven days after transplantation, incisional wounds were made in the skin grafts. In the first phase of the study, wounds were harvested from 1 hour to 4 weeks postwounding, and immunohistochemistry was performed for TGF beta (isoform nonspecific), TGF beta 1, and TGF beta 2. Scarfree wounds in the fetal skin grafts did not show TGF beta staining. In contrast, wounds in adult grafts that heal with scar demonstrated isoform nonspecific TGF beta staining from 6 hours through 21 days, TGF beta 1 from 6 hours through 21 days, and TGF beta 2 from 12 hours through 7 days. In the second phase of the study, a slow-release disk with 0.01, 0.1, 1.0, or 10 micrograms of TGF beta 1 was placed beneath the fetal skin graft at the time of wounding. Fourteen days postwounding, there was marked scarring in the fetal grafts treated with TGF beta 1, and the size of the scar was proportional to the amount of TGF beta 1 applied.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired spinal cord injury in human fetuses with myelomeningocele.

Experimental studies have shown that there is a potential to attempt in utero repair of myelomeningocele in human fetuses. To provide a better understanding of the pathology of these lesions we prospectively studied eight stillborn human fetuses with myelomeningocele autopsied at The Johns Hopkins Hospital. The intact vertebral column with surrounding structures was removed, processed as a single block, and prepared as serial histologic sections. Study of the slides showed in all cases that in the center of the myelomeningocele the vertebral arch was open, the arrangement of meninges was such that the dura mater was open and in continuity with the deep layers of the dermis, and the pia mater was open and in continuity with a layer consisting of the superficial dermis and the epidermis. These meningeal relationships created an abnormally configured arachnoid space containing cerebrospinal fluid ventral to the spinal cord, which rested on the open pia mater and was exposed on the dorsal aspect of the sac. At the level of the myelomeningocele the naked cord had undergone varying degrees of injury up to complete loss of neural tissue. Where ventral remnants of the cord remained it was evident that a large degree of normal development of the cord had occurred. In most instances it appeared that the injury or destruction of the dorsal spinal cord was recent and consistent with occurrence during delivery. The results of this study support the concept that in utero surgery could preserve and protect the exposed spinal cord in a myelomeningocele of a human fetus and thus could reduce the severity of the neurologic deficit at birth.

Markers to Evaluate the Quality and Self-Renewing Potential of Engineered Human Skin Substitutes In Vitro and after Transplantation

We screened a series of antibodies for their exclusive binding to the human hair follicle bulge. In a second step these antibodies were to be used to identify basal keratinocytes and potential epithelial stem cells in the human epidermis and in engineered skin substitutes. Of all the antibodies screened, we identified only one, designated C8/144B, that exclusively recognized the hair follicle bulge. However, C8/144B-binding cells were never detected in the human epidermal stratum basale. In the bulge C8/144B-binding cells gave rise to cytokeratin 19-positive cells, which were also tracked in the outer root sheath between bulge and the hair follicle matrix. Remarkably, cytokeratin 19-expressing cells were never detected in the hair follicle infundibulum. Yet, cytokeratin 19-expressing keratinocytes were found in the epidermal stratum basale of normal skin as a subpopulation of cytokeratin 15-positive (not C8/144B-positive) basal keratinocytes. Cytokeratin 19/cytokeratin 15-positive keratinocytes decreased significantly with age. We suggest that cytokeratin 19-expressing cells represent a subpopulation of basal keratinocytes in neonates and young children (up to 1.5 years) that is particularly adapted to the lateral expansion of growing skin. Our data show that cytokeratin 19 in combination with cytokeratin 15 is an important marker to routinely monitor epidermal homeostasis and (at least indirectly) the self-renewing potential of engineered skin.

Cross-linking of the dermo-epidermal junction of skin regenerating from keratinocyte autografts. Anchoring fibrils are a target for tissue transglutaminase

Since transglutaminases create covalent gamma-glutamyl-epsilon-lysine cross-links between extracellular matrix proteins they are prime candidates for stabilizing tissue during wound healing. Therefore, we studied the temporo-spatial expression of transglutaminase activity in skin regenerating from cultured epithelial autografts in severely burned children by the specific incorporation of monodansylcadaverine into cryostat sections from skin biopsies obtained between 5 d to 17 mo after grafting. The dansyl label was subsequently immunolocalized in the epidermis, dermal connective tissue, and along the basement membrane. Incubation of cryosections of normal and regenerating skin with purified tissue transglutaminase confirmed the dermo-epidermal junction and the papillary dermis as targets for this enzyme and revealed that in regenerating skin transamidation of the basement membrane zone was completed only 4-5 mo after grafting. Immunoelectron microscopy revealed that three distinct regions on the central portion of anchoring fibrils were positive for monodansylcadaverine in normal skin which were negative during the initial phase of de novo formation of anchoring fibrils in regenerating skin. Biochemically, we identified collagen VII as potential substrate for tissue transglutaminase. Thus, tissue transglutaminase appears to play an important role not only in cross-linking of the papillary dermis but also of the dermo-epidermal junction in particular.

Matriderm versus Integra: a comparative experimental study.

AIM To compare engraftment rates and vascularisation in a rat model using either Integra Artificial Skin or Matriderm. METHODS Matriderm and the dermal part of Integra were compared in a two-step procedure including matrix implantation and subsequent epidermal grafting. Neonatal rat epidermis was used as coverage to test for rapid and complete take. RESULTS Efficiency and quality of vascularisation expressed by take rate of epidermis, and thickness of resulting neodermis, were identical for both matrices. CONCLUSION This first comparison of Matriderm with Integra in a rat model revealed no major differences in engraftment rates or vascularisation.

Tissue-engineered dermo-epidermal skin grafts prevascularized with adipose-derived cells.

The major problem in skin grafting is that tissue-engineered skin grafts after their transplantation are initially entirely dependent on diffusion. Since this process is slow and inefficient, nutrients, growth factors, and oxygen will insufficiently be supplied and the regenerating graft will undergo a physiological crisis, resulting in scar-like dermal structures and shrinkage. The tissue-engineering of a vascular network in human dermo-epidermal skin substitutes (DESS) is a promising approach to overcome this limitation. Here we report, for the first time, on the use of the adipose stromal vascular fraction (SVF)-derived endothelial cell population to tissue-engineer DESS containing a highly efficient capillary plexus. To develop vascular networks in vitro, we employed optimized 3D fibrin or collagen type I hydrogel systems. Upon transplantation onto immune-deficient rats, these pre-formed vascular networks anastomosed to the recipients vasculature within only four days. As a consequence, the neo-epidermis efficiently established tissue homeostasis, the dermis underwent almost no contraction, and showed sustained epidermal coverage in vivo. Overall, the here described rapid and efficient perfusion of SVF-based skin grafts opens new perspectives for the treatment of hitherto unmet clinical needs in burn/plastic surgery and dermatology.

Bioengineering Dermo-Epidermal Skin Grafts with Blood and Lymphatic Capillaries

Human lymphatic capillaries were engineered in a 3D hydrogel system to improve dermo-epidermal skin grafting. Skin Grafts Need Plumbing, Too To help heal a severe burn or wound, clinicians surgically transplant skin grafts, which consist of the epidermis (outer skin layer) and, often, part of the dermis (deeper layer, directly below the epidermis). The success of these grafts, however, is limited by the ability of blood vessels to form in the newly transplanted skin and deliver nutrients to the cells. Research has also suggested that the lymphatics may be necessary for skin graft survival, by essentially draining immune cells, debris, and excess fluid from the wounded area. Here, Marino, Luginbühl, and colleagues engineered a skin graft that wasn’t just the patient’s skin cells—it also contained both lymph and blood capillaries “prevascularized” ex vivo and then transplanted onto a wound. The authors created the dermo-epidermal skin grafts by taking cells from human foreskin, called human dermal microvascular endothelial cells (HDMECs), and embedding them in three-dimensional hydrogels. HDMECs consist of a mixture of both lymphatic endothelial cells and blood vessel endothelial cells, so both types of functional capillaries—blood and lymph—formed from these cells in vitro in the fibrin or collagen hydrogels. Moving in vivo, the authors transplanted engineered skin grafts containing the HDMECs as well as human fibroblasts and keratinocyes—two cell types found in skin—onto the wounded backs of nude rats (animals without an immune system). Marino, Luginbühl, et al. reported that the human skin grafts formed the expected skin layers after 2 weeks, connected with existing rat lymphatic capillaries, and drained fluid away from the wound. Although testing and characterization are still needed in animals with an immune system and with skin similar to humans (such as a pig), these engineered dermo-epidermal hydrogels potentially represent the next generation of skin grafts, complete with the vascular and lymphatic plumbing and ready to transplant. The first bioengineered, autologous, dermo-epidermal skin grafts are presently undergoing clinical trials; hence, it is reasonable to envisage the next clinical step at the forefront of plastic and burn surgery, which is the generation of autologous skin grafts that contain vascular plexuses, preformed in vitro. As the importance of the blood, and particularly the lymphatic vascular system, is increasingly recognized, it is attractive to engineer both human blood and lymphatic vessels in one tissue or organ graft. We show here that functional lymphatic capillaries can be generated using three-dimensional hydrogels. Like normal lymphatics, these capillaries branch, form lumen, and take up fluid in vitro and in vivo after transplantation onto immunocompromised rodents. Formation of lymphatic capillaries could be modulated by both lymphangiogenic and anti-lymphangiogenic stimuli, demonstrating the potential usefulness of this system for in vitro testing. Blood and lymphatic endothelial cells never intermixed during vessel development, nor did blood and lymphatic capillaries anastomose under the described circumstances. After transplantation of the engineered grafts, the human lymphatic capillaries anastomosed to the nude rat’s lymphatic plexus and supported fluid drainage. Successful preclinical results suggest that these skin grafts could be applied on patients suffering from severe skin defects.

Physiologically low oxygen concentrations in fetal skin regulate hypoxia-inducible factor 1 and transforming growth factor-beta3.

In the first‐trimester mammalian fetus, skin wounds heal with perfect reconstitution of the dermal architecture without scar formation. Understanding environmental molecular regulation in fetal wound healing may reveal scar‐limiting therapeutical strategies for the prevention of postnatal scarring wound repair. Therefore, we performed studies on fetal skin oxygenation and skin and wound expression of hypoxia‐inducible factor 1α (HIF‐1α) in the sheep model in vivo and performed studies on the potential relevance of HIF‐1α during wound healing in vitro. Skin oxygen partial pressure levels were hypoxic throughout normal development. In nonscarring fetal skin at gestation day (GD)60, HIF‐1 α could be detected neither in healthy nor in wounded tissue. At GD100, in wounds with minimal scar formation, HIF‐1 α was expressed in fibroblasts and was markedly up‐regulated at the wound edge. In scarring fetal wounds at GD120, HIF‐1 α was predominantly expressed in inflammatory cells. Expression of transforming growth factor β3 (TGF‐β3), a potent antiscarring cytokine, overlapped with HIF‐1 α expression at GD100. HIF‐1 α‐deficient mouse embryonic fibroblasts showed impaired migratory capabilities and demonstrated that TGF‐β3, but not proscarring TGF‐β1, manifests hypoxia‐ and HIF‐1α‐dependent regulation. In conclusion, HIF‐1‐dependent regulation of a potent antiscarring cytokine may provide new strategies for antiscarring manipulation of wound healing.