Claudia Meuli-Simmen

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.

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.

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.

Functional and morphometric evaluation of end-to-side neurorrhaphy for muscle reinnervation.

This study was undertaken to quantify the effect of motor collateral sprouting in an end-to-side repair model allowing end organ contact. Besides documentation of the functional outcome of muscle reinnervation by end-to-side neurorrhaphy, this experimental work was performed to determine possible downgrading effects to the donor nerve at end organ level. In 24 female New Zealand White rabbits, the motor nerve branch to the rectus femoris muscle of the right hindlimb was dissected, cut, and sutured end-to-side to the motor branch to the vastus medialis muscle after creating an epineural window. The 24 rabbits were divided into two groups of 12 each, with the second group receiving additional crush injury of the vastus branch. After a period of 8 months, maximum tetanic tension in the reinnervated rectus femoris and the vastus medialis muscles was determined. The contralateral healthy side served as control. The reinnervated rectus femoris muscle showed an average maximum tetanic force of 24.9 N (control 26.2 N, p = 0.7827), and the donor vastus medialis muscle 11.0 N (control 7.3 N, p = 0.0223). There were no statistically significant differences between the two experimental groups (p = 0.9914). The average number of regenerated myelinated nerve fibers in the rectus femoris motor branch was 1185 ± 342 (control, 806 ± 166), and the mean diameter was 4.6 ± 0.6 &mgr;m (control, 9.4 ± 1.0 &mgr;m). In the motor branch to the vastus medialis muscle, the mean fiber number proximal to the coaptation site was 1227 (±441), and decreased distal to the coaptation site to 795 (±270). The average difference of axon counts in the donor nerve proximal to distal regarding the repair site was 483.7 ± 264.2. In the contralateral motor branch to the vastus medialis muscle, 540 (±175) myelinated nerve fibers were counted. In nearly all cross-section specimens of the motor branch to the vastus medialis muscle, altered nerve fibers could be identified in one fascicle distal and proximal to the repair site. The results show a relevant functional reinnervation by end-to-side neurorrhaphy without functional impairment of the donor muscle. It seems to be evident that most axons in the attached segment were derived from collateral sprouts. Nonetheless, the present study confirms that end-to-side neurorrhaphy is a reliable method of reconstruction for damaged nerves, which should be applied clinically in a more extended manner. (Plast. Reconstr. Surg. 106: 383, 2000.)

Breast augmentation and reconstructive surgery: MR imaging of implant rupture and malignancy

Abstract. The purpose of this study was to assess the diagnostic accuracy of MRI in detecting prosthesis integrity and malignancy after breast augmentation and reconstruction. Forty-one implants in 25 patients were analyzed by MRI before surgical removal. Imaging results were compared with ex vivo findings. Magnetic resonance imaging of the breast was performed on a 1.5-T system using a dedicated surface breast coil. Axial and sagittal T2-weighted fast spin-echo as well as dynamic contrast-enhanced T1-weighted gradient-recalled-echo sequences were acquired. The linguine sign indicating collapse of the silicone shell or siliconomas indicating free silicone proved implant rupture, whereas early focal contrast enhancement of a lesion was suspicious for malignancy. The sensitivity for detection of implant rupture was 86.7% with a specificity of 88.5%. The positive and negative predictive values were 81.3 and 92.0%, respectively. The linguine sign as a predictor of intracapsular implant rupture had a sensitivity of 80% with a specificity of 96.2%. Magnetic resonance imaging revealed two lesions with suspicious contrast enhancement (one carcinoma, one extra-abdominal fibromatosis). Magnetic resonance imaging is a reliable and reproducible technique for diagnosing both implant rupture and malignant lesions in women after breast augmentation and reconstruction.

Efficacy of cultured epithelial autografts in pediatric burns and reconstructive surgery

BACKGROUND Cultured epithelial autografts are regularly used in burn patients, but they have not been tested in patients undergoing reconstructive surgery. The aim of this study was to analyze and compare the efficacy of cultured grafts in both burn and reconstructive surgery patients. METHODS In six children with severe and massive burns, full-thickness areas were grafted with cultured grafts. In another six children with hypertrophic or hyperpigmented scars, or both, cultured grafts were used to cover defects resulting from scar excision or deep dermabrasion. RESULTS In burn surgery the final cover rate averaged 60% (range, 0% to 100%). The functional and cosmetic results were good and at least equivalent to results after conventional grafting. Fragility, infection, and, in particular, mechanical instability of cultured grafts during the first weeks after transplantation were the main problems encountered. In reconstructive surgery the final cover rate was 100% in all patients. The functional and cosmetic results were very good and considered better than those obtained by using conventional grafting techniques. No major management problems were encountered. CONCLUSIONS In massively burned children, cultured epithelial autografts represent an effective additional and potentially lifesaving method to conventional grafting. Questions remain regarding the use of this technique to treat less severe burns. For resurfacing-type scar revisions, cultured epithelial autografts yield excellent results that appear to be superior to those of conventional techniques.

Gene Expression along the Cerebral- Spinal Axis after Regional Gene Delivery

We demonstrate here that intracerebroventricular or spinal cord (intrathecal) injection of either plasmid DNA alone or cationic liposome: DNA complexes (CLDCs) produces significant levels of expression of both reporter genes and biologically relevant genes in nonparenchymal cells lining both the brain and the spinal cord. Gene expression was identified both within the spinal cord and the brain after intracerebroventricular or intrathecal injection of either CLDCs or plasmid DNA alone. Intracerebroventricular or intrathecal injection of CLDCs containing the beta-galactosidase (beta-Gal) gene produced patchy, widely scattered areas of beta-Gal expression. The chloramphenicol acetyltransferase (CAT) reporter gene product reached peak levels between 24 hr and 1 week postinjection, and was still present at significant levels 3 weeks after a single intracerebroventricular or intrathecal injection. Intrathecal injection of the human granulocyte colony-stimulating factor (G-CSF) gene produced high levels of hG-CSF activity in both the spinal cord and the brain. Intracerebroventricular injection of CLDCs containing the murine nerve growth factor (NGF) gene increased mNGF levels in the hippocampus, a target region for cholinergic neurons in the medial septum, and increased cholinergic neurotransmitter synthetic enzyme choline acetyltransferase (ChAT) activity within the brain, a well-characterized effect of both purified and recombinant NGF protein. These findings indicate that intracerebroventricular or intrathecal injection of CLDCs can produce significant levels of expression of biologically and therapeutically relevant genes within the CNS. Efficient gene transfer into the CNS will facilitate the evaluation of gene function and regulation within the brain and spinal cord. We attempted to transfer and express genes within the brain and spinal cord by direct CNS injection of either DNA alone or CLDCs into the intraventricular and subarachnoid compartments. We show that intracerebroventricular or spinal cord (intrathecal) injection of either plasmid DNA alone or CLDCs produces significant levels of expression of both reporter genes and biologically relevant genes in nonparenchymal cells lining both the brain and the spinal cord. Intrathecal injection of the hG-CSF gene produced high levels of hG-CSF activity in both the spinal cord and the brain. Intracerebroventricular injection of CLDCs containing the murine NGF gene increased mNGF levels in the hippocampus, and increased cholinergic neurotransmitter synthetic enzyme ChAT activity within the brain. Locoregional diffusion of gene products expressed by transfected meningeal lining cells into brain and spinal cord parenchyma could potentially target secreted proteins within brain and spinal cord regions relevant to neuropathological states while limiting peripheral side effects.

FETAL SURGERY FOR MYELOMENINGOCELE: PANACEA OR PERIL?

Myelomeningocele affects thousands of children worldwide with devastating consequences. In an effort to improve neurologic outcome, fetal surgery has been performed for myelomeningocele for the past 5 years. Sensorimotor function is not appreciably improved, although there may be a reduction in hindbrain herniation and a decreased need for ventriculoperitoneal shunting. The long-term clinical consequences of these findings are not clear. What is clear, however, is that further study in the form of a prospective, randomized trial is mandatory.

Human Eccrine Sweat Gland Cells Turn into Melanin-Uptaking Keratinocytes in Dermo-Epidermal Skin Substitutes

Recently, Biedermann et al. (2010) have demonstrated that human eccrine sweat gland cells can develop a multilayered epidermis. The question still remains whether these cells can fulfill exclusive and very specific functional properties of epidermal keratinocytes, such as the incorporation of melanin, a feature absent in sweat gland cells. We added human melanocytes to eccrine sweat gland cells to let them develop into an epidermal analog in vivo. The interaction between melanocytes and sweat gland-derived keratinocytes was investigated. The following results were gained: (1) macroscopically, a pigmentation of the substitutes was seen 2-3 weeks after transplantation; (2) we confirmed the development of a multilayered, stratified epidermis with melanocytes distributed evenly throughout the basal layer; (3) melanocytic dendrites projected to suprabasal layers; and (4) melanin was observed to be integrated into former eccrine sweat gland cells. These skin substitutes were similar or equal to skin substitutes cultured from human epidermal keratinocytes. The only differences observed were a delay in pigmentation and less melanin uptake. These data suggest that eccrine sweat gland cells can form a functional epidermal melanin unit, thereby providing striking evidence that they can assume one of the most characteristic keratinocyte properties.