The impact of genital trauma on wounded servicemen: qualitative study.

BACKGROUND: Recent military operations in Afghanistan have resulted in considerable lower limb trauma. Associated with this trauma have been serious injuries to the genitalia. No previous study has looked at the effect of traumatic genital injury on the individual. This study expresses the impact of these injuries. METHODS: A qualitative evaluation, interviewing 13 male patients with extensive genital injuries, including 11 patients with orchidectomies. RESULTS: Patients attach a very high importance to their genital injury, rating this injury as having more impact than lower limb amputations. They also have a high expectation for the recovery of sexual function. Psychological outcomes are better when an individual's future fertility is known at an early stage. CONCLUSION: Better outcomes are achieved for the patient when psychological and surgical support if provided throughout the long treatment and recovery process It is important that every effort is made to preserve fertility right from the point of injury.

Stem cells for mesothelial repair: an understudied modality.

Adhesions are bands of fibrous tissue that form between opposing organs and the peritoneum, restricting vital intrapleural and abdominal movement. They remain a major problem in abdominal surgery, occurring in more than three fourths of patients following laparotomy. Adhesions result when injury to the mesothelium is not repaired by mesothelial cells and can be viewed as scar tissue formation. The mechanism of mesothelial healing suggested the involvement of stem cells in the process. It has long been known that peritoneal wounds heal in the same amount of time regardless of size. Therefore, the mesothelium could not regenerate solely by proliferation and centripetal migration of cells at the wound edge as occurs in the healing of skin epithelium. Several studies suggest the presence of i) mesothelial stem cells that can differentiate into mesothelial cells and a few other phenotypes and/or ii) that mesothelial cells are themselves stem cells. Other studies have suggested that adult stem cells in the muscle underlying the peritoneum can differentiate into mesothelial cells and contribute to healing. Prevention of abdominal adhesions have been accomplished by delivery of autologous mesothelial cells and multipotent adult stem cells isolated from skeletal muscle. Adult stem cells from sources other than the serosal tissue offer an alternative treatment modality to prevent the formation of abdominal adhesions.

Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors.

This study details the profile of 13 cell surface cluster differentiation markers on human reserve stem cells derived from connective tissues. Stem cells were isolated from the connective tissues of dermis and skeletal muscle derived from fetal, mature, and geriatric humans. An insulin/dexamethasone phenotypic bioassay was used to determine the identity of the stem cells from each population. All populations contained lineage-committed myogenic, adipogenic, chondrogenic, and osteogenic progenitor stem cells as well as lineage-uncommitted pluripotent stem cells capable of forming muscle, adipocytes, cartilage, bone, fibroblasts, and endothelial cells. Flow cytometric analysis of adult stem cell populations revealed positive staining for CD34 and CD90 and negative staining for CD3, CD4, CD8, CD11c, CD33, CD36, CD38, CD45, CD117, Glycophorin-A, and HLA DR-II.

Bioactive factors affect proliferation and phenotypic expression in progenitor and pluripotent stem cells.

Progenitor and pluripotent stem cells reside within connective tissue compartments. They are also present in granulation tissue. This study examined the effects of treating these two cell populations with eight bioactive factors. Cells were assayed for DNA content as a measure of proliferation and for tissue-specific phenotypic markers as measures of lineage progression and lineage commitment. Platelet-derived endothelial growth factor and insulin-like growth factor-II did not induce proliferation in either population. However, dexamethasone, insulin, insulin-like growth factor-I, muscle morphogenetic protein, platelet-derived growth factor-AA, and platelet-derived growth factor-BB stimulated proliferation in one or both cell populations. Platelet-derived growth factor-BB was the most potent stimulator of proliferation in either population. Phenotypic expression markers were induced in the progenitor cells by insulin, insulin-like growth factor-I, insulin-like growth factor-II, dexamethasone, and muscle morphogenetic protein. However, only dexamethasone and muscle morphogenetic protein induced phenotypic expression markers in the pluripotent cells. Platelet-derived endothelial cell growth factor, platelet-derived growth factor-AA, and platelet-derived growth factor-BB did not induce phenotypic expression markers in progenitor or pluripotent cells. This study suggests the potential for using progenitor and pluripotent cells as an in vitro model to ascertain the effects of various bioactive factors on stem cells potentially involved in tissue maintenance and repair.

Muscle morphogenetic protein induces myogenic gene expression in Swiss-3T3 cells.

Myogenesis is thought to be regulated by the MyoD family of regulatory genes, which includes MyoD, myogenin, MRF- 4/myf-6, and myf-5. In situ hybridization studies of vertebrate skeletal muscle development have shown the colocalization of the MyoD family of regulatory genes to specific stages of muscle development. Although many studies have analyzed the regulatory role of these genes during myogenesis, there have been few reports dealing with the activation of these myogenic regulatory genes by exogenous agents. We have previously shown that muscle morphogenetic protein induces myogenesis in clonal populations of avian pluripotent stem cells. The current study was designed to examine the ability of muscle morphogenetic protein to induce myogenesis in a clonal population derived from the established fibroblastic Swiss-3T3 cell line. Swiss-3T3 cells were cloned to generate separate cell populations, tested for pluripotency, propagated through 690 cell doublings, retested for pluripotency, treated with muscle morphogenetic protein, and examined for the induction of gene expression using probes for the transcription products of MyoD and myogenin. Muscle morphogenetic protein induced the expression of mRNAs for MyoD and myogenin, suggesting a role for this compound as an exogenous activator of myogenesis.

Interrogative suggestibility in patients with conversion disorders.

We tested the hypothesis that increased interrogative suggestibility may contribute to the shaping and maintaining of conversions symptoms. Interrogative suggestibility was measured in 12 patients with conversion disorder and 10 control patients with confirmed neurological disease matched for age, premorbid intelligence, and as closely as possible in terms of their neurological symptoms to the patients with conversion disorder. Our observations do not support the contention that individual differences in interrogative suggestibility are of importance in the etiology of conversion disorders.

Formation of abdominal adhesions is inhibited by antibodies to transforming growth factor-beta1.

Transforming growth factor-beta (TGF-beta) is an important factor in regulating the inflammatory response and the production of extracellular matrix by fibroblasts. These two processes are linked in the formation of fibrous adhesions after abdominal surgery. When the mesothelium is injured a fibrin strand is produced which is populated first by inflammatory cells then by fibroblasts which secrete extracellular matrix forming a permanent adhesion. TGF-beta promotes both chemotaxis of monocytes and the production of extracellular matrix by fibroblasts. We have used a model of abdominal adhesions in rats in which a circle of peritoneum is dissected and then sutured into place again. After 2 weeks the rats are euthanized and the adhesions are scored. Six groups of 10 rats each underwent this surgery. Group I served as the operative control. Group II was treated with saline which was injected immediately after surgery and on Days 1 and 2 after surgery (vehicle control). Using the same protocol with saline as vehicle, the other four groups of rats were treated with nonspecific IgG (150 microgram per day), anti-TGF-beta (panspecific, 167 microgram per day), anti-TGF-beta1 (67 microgram per day), or anti-TGF-beta2 (50 microgram per day). The rats injected with anti-TGF-beta1 had significantly lower adhesion scores (P < 0.05) than the controls. Rats injected with anti-TGF-beta2 or anti-TGF-beta (panspecific) did not differ significantly from the control saline-injected rats. The results indicate that specifically reducing levels of TGF-beta1 alone can be effective in preventing abdominal adhesions.

Recombinant human bone morphogenetic proteins-2 and -4 induce several mesenchymal phenotypes in culture.

Bone morphogenetic protein has previously been shown to induce the formation of cartilage and bone in vivo. We have isolated a population of mesenchymal stem cells from rat skeletal muscle capable of forming multiple mesodermal morphologies in vitro. These cells were treated with recombinant human bone morphogenetic proteins-2 and -4 to determine the differentiation-inducing activities of bone morphogenetic protein on these cells. The mesenchymal stem cells were cultured in medium with 10% preselected horse serum containing 0 to 100 ng/ml recombinant human bone morphogenetic proteins-2 or -4 for a maximum of 4 weeks. Control cultures maintained the stellate morphology of mesenchymal stem cells. Cultures treated with recombinant human bone morphogenetic protein-2 exhibited discrete cartilage nodules and mineralized bone nodules. The first increase in chondrogenesis was seen at 0.5 ng/ml. Cultures treated with recombinant human bone morphogenetic protein-4 also exhibited an increase in chondrogenesis at the higher concentration of 2 ng/ml. Skeletal myotubes and adipocytes also appeared in cultures treated with either bone morphogenetic protein. Mesenchymal stem cells do respond to inductive factors, but bone morphogenetic proteins-2 and -4 were not specific for the induction of cartilage and bone.

Effect of rat mesenchymal stem cells on development of abdominal adhesions after surgery.

One of the common and most serious side effects of abdominal surgery is the formation of adhesions within the peritoneal cavity during healing. Efforts to prevent adhesion formation have concentrated on inhibiting the inflammatory response, inhibiting the formation or encouraging the lysis of fibrin, and protection of the damaged serosal surface. We are interested in regenerating the serosal surface by providing a source of mesothelial progenitor cells. Rats were divided into groups of 10 each. Abdominal adhesions were created by removing a circle of peritoneum and suturing it back into place. Two weeks later the rats were euthanized and the adhesions scored on a scale of 0-5. A population of mesenchymal stem cells (MSCs) isolated from the skeletal muscle of neonatal rats was tested. The cells were grown in primary culture to expand the population and then trypsinized and frozen at -80 degrees C. They are then thawed and grown in secondary culture before use. The control group were injected with saline i.p. immediately after surgery. The experimental groups received (1) 1.4 X 10(6) MSCs, (2) 5 X 10(6) MSCs, (3) 7.5 X 10(6) dead MSCs, (4) 5 X 10(6) rat smooth muscle cells immediately post-op, and (5) 5 X 10(6) MSCs 4-6 hours after surgery. Only live MSCs given immediately after surgery by i.p. injection significantly decreased the adhesion scores of the rats (mean score of 3.5 vs 0.9). MSCs injected i.p. 4-6 hours after surgery actually increased the adhesion scores (3.5 vs 4.7), and rat smooth muscle cells injected i.p. immediately after surgery had no effect on adhesions. The exact mechanism of action of the MSCs is unknown at this time. However, we postulate that the MSCs have the capacity to differentiate into mesothelial cells capable of repopulating the injured mesothelium.

A population of cells isolated from rat heart capable of differentiating into several mesodermal phenotypes.

A population of stem cells has been isolated from embryonic avian and neonatal rat skeletal muscle. These cells differentiate into several mesodermal phenotypes in culture upon treatment with dexamethasone. This study reports the isolation of a similar population of stem cells from another mesodermal tissue, the heart. Hearts were excised from 3- to 5-day- old rats, minced, and treated with a collagenase-dispase solution. Single cells were collected by centrifugation, washed, and plated in dishes. The cells were grown to confluence, trypsinized, and frozen at -80 degrees C in 7.5% dimethylsulfoxide. After at least 24 hr, the cells were thawed and plated in 24-well plates and treated with media containing dexamethasone at concentrations of 10(-6)-10(-10) M for 4 weeks. Control cultures contained mononucleated cells with a stellate morphology. Treatment with dexamethasone resulted in the appearance of several mesodermal phenotypes. Bone and cartilage nodules were identified with von Kossa and Alcian blue staining respectively. Adipocytes were identified using Sudan black B stain. Smooth muscle cells were identified by an anti-smooth muscle alpha-actin antibody, and skeletal myotubes were stained with anti-myosin antibody. Large binuclear cells with obvious fibers were noted and stained with anti-desmin. These binuclear cells appeared in both the control and the dexamethasone-treated cultures and were tentatively identified as cardiomyocytes. These data strongly suggest the existence of a population of mesenchymal stem cells in neonatal rat heart.

Loin pain and haematuria syndrome: a somatoform disorder.

Fifteen patients with the loin pain and haematuria syndrome (LPH) were compared with 10 patients with complicated renal stone disease referred to the same tertiary centre and matched for age, sex and duration of illness. LPH patients had a history of three times more medically unexplained somatic symptoms other than loin pain (p < 0.01) and a higher proportion took analgesics regularly (p < 0.01). The onset of pain was associated with an adverse psychologically important life-event in eight of the LPH patients but in none of the controls (p < 0.02). LPH patients more frequently recalled serious parental illness and disability in childhood (p < 0.001) than controls, and a higher proportion felt responsible for causing or alleviating parental illness or distress (p < 0.05). LPH subjects scored higher in the 'paternal care' dimension of the Parental Bonding Instrument (p < 0.05). No difference was found between LPH patients and controls in terms of current depression and anxiety but both groups exhibited high rates of lifetime depression. LPH patients expressed lower levels of anger and hostility (p < 0.002) than did controls. Our observations suggest that psychological factors are of major importance in the aetiology of LPH, which may represent a type of somatoform pain disorder.

A population of cells resident within embryonic and newborn rat skeletal muscle is capable of differentiating into multiple mesodermal phenotypes.

We have previously shown a population of putative mesenchymal stem cells in the connective tissue surrounding embryonic avian skeletal muscle. These cells differentiate into at least five recognizable phenotypes in culture: fibroblasts, chondrocytes, myotubes, osteoblasts, and adipocytes. We have now isolated a similar population of cells from fetal and newborn rat skeletal muscle. Cells from rat leg muscle were dissected, minced, and then enzymatically digested with a collagenase-dispase solution. The dissociated cells were plated and allowed to differentiate into two recognizable populations: myotubes and stellate mononucleated cells. The cells were then trypsinized, filtered through a 20 microm filter to remove the myotubes, frozen at -80 degrees C, then thawed and replated. In culture the cells maintained their stellate structure. However, under treatment with dexamethasone, a nonspecific differentiating agent, seven morphologic conditions emerged: cells with refractile vesicles that stained with Sudan black B (adipocytes), multinucleated cells that spontaneously contracted in culture and stained with an antibody to myosin (myotubes), round cells whose extracellular matrix stained with Alcian blue, pH 1.0 (chondrocytes), polygonal cells whose extracellular matrix stained with Von Kossa's stain (osteoblasts), cells with filaments that stained with an antibody to smooth muscle a-actin (smooth muscle cells), cells that incorporated acetylated low density lipoprotein (endothelial cells), and spindle-shaped cells that grew in a swirl pattern (fibroblasts). The initial population is tentatively classified as putative mesenchymal stem cells. The presence of these cells point to the existence of stem cells in the postembryonic mammal that could provide a basis for tissue regeneration as opposed to scar tissue formation during wound healing.

Inhibition of epidural scar formation after lumbar laminectomy in the rat.

STUDY DESIGN: An animal model of laminectomy in rats was used to study scar tissue formation around the spinal cord. Dexamethasone, in controlled-release form, was tested in this system for its ability to decrease fibrous tissue formation. OBJECTIVES: The results were evaluated to determine whether dexamethasone in a biodegradable controlled-release vehicle could be used to limit scar tissue formation around the spinal cord after laminectomy. SUMMARY OF BACKGROUND DATA: Steroids can delay the formation of scar tissue. Continued treatment with dexamethasone results in various unacceptable side effects. Use of biodegradable controlled-release vehicles to deliver drugs may allow for prolonged low-dose treatment, concentrated at the surgical site, thereby avoiding side effects. METHODS: Forty-four Sprague Dawley rats underwent laminectomies and were treated with dexamethasone in one of two controlled-release vehicles or with vehicle alone. After 4 weeks, the rats were killed and histologic sections prepared from the spines were examined and graded by a pathologist. In addition, the dexamethasone preparations were introduced into Hunt-Schilling wound chambers, which were implanted in rats. Four weeks after implantation, the wound chambers were removed and the tissue inside was assayed for DNA and protein content. RESULTS: Dexamethasone acetate (Decadron, MSD, West Point, PA) significantly reduced the density of the scar tissue undermining the laminas. Steroids embedded in polymer did not change the scar formation in the back, but did decrease protein and DNA values in wound chamber tissues. CONCLUSIONS: Long-term release of small amounts of steroid from the polymer poly-carboxy-phenoxypropane does not appear to reduce scar at laminectomy sites but does decrease the protein:DNA ratio in wound chambers. In contrast, Decadron does not significantly alter the biochemistry of wound chamber tissue but does reduce scar in the back.

Differentiation factors induce expression of muscle, fat, cartilage, and bone in a clone of mouse pluripotent mesenchymal stem cells.

Growth factors have been used experimentally to accelerate wound healing by increasing scar tissue formation at a wound site. These studies suggest that stimulation of fibroblastic differentiation and proliferation are essential components of adult tissue repair. Recent studies report the presence of mesenchymal stem cells within granulation tissue and as connective tissue-resident stem cells. This suggests that mesenchymal stem cells as well as fibroblasts may contribute to wound healing and repair. To determine the potential for mesenchymal stem cells to contribute to nonfibrogenic tissue repair, a clonal population of murine mesenchymal stem cells was examined with dexamethasone, a general differentiation agent, and muscle morphogenetic protein, a specific differentiation-inducing agent. Dexamethasone induced the expression of phenotypic markers for fat, cartilage, and bone in the stem cells. Muscle morphogenetic protein induced the expression of mRNAs for the muscle specific regulatory genes MyoD1 and myogenin in these cells. These results suggest that pluripotent mesenchymal stem cells within connective tissue compartments and granulation tissue have the potential to contribute to functional tissue restoration, rather than contributing solely to fibrogenic scar tissue formation during tissue repair.

Mesenchymal stem cells reside within the connective tissues of many organs.

Previous studies have noted the presence of mesenchymal stem cells located within the connective tissue matrices of avian skeletal muscle, dermis, and heart. In these studies, clonal analysis coupled with dexamethasone treatment revealed the presence of multiple populations of stem cells composed of both lineage-committed progenitor mesenchymal stem cells and lineage-uncommitted pluripotent mesenchymal stem cells. The present study was undertaken to assess the distribution of these stem cells in the connective tissues throughout various regions of the body. Day 11 chick embryos were divided into 26 separate regions. Heart, limb skeletal muscle, and limb dermis were included as control tissues. Cells were harvested enzymatically and grown using conditions optimal for the isolation, cryopreservation, and propagation of avian mesenchymal stem cells. Cell aliquots were plated, incubated with various concentrations of dexamethasone, and examined for differentiated phenotypes. Four recurring phenotypes appeared in dexamethasone-treated stem cells: skeletal muscle myotubes, fat cells, cartilage nodules, and bone nodules. These results suggest that progenitor mesenchymal stem cells and putative pluripotent mesenchymal stem cells with the potential to form at least four tissues of mesodermal origin have a widespread distribution throughout the body, being located within the connective tissue compartments of many organs and organ systems.

Repair of articular cartilage defects using mesenchymal stem cells.

Degeneration of articular cartilage in osteoarthritis is a serious medical problem. We have isolated a population of cells from the connective tissue of mammals termed mesenchymal stem cells (MSCs) for their apparent unlimited growth potential and their ability to differentiate into several phenotypes of the mesodermal lineage, including cartilage and bone. These qualities make them ideal candidates for cartilage repair. We isolated MSCs from adult rabbit muscle and cultured them in vitro into porous polyglycolic acid polymer matrices. The matrices were implanted into 3-mm-diameter full thickness defects in rabbit knees with empty polymer matrices serving as the contralateral controls. The implants were harvested 6 and 12 weeks postop. At 6 weeks, the controls contained fibrocartilage while the experimentals seemed to contain undifferentiated cells. By 12 weeks postop, the controls contained limited fibrocartilage and extensive connective tissue, but no subchondral bone. In contrast, the implants containing MSCs had a surface layer of cartilage approximately the same thickness as normal articular cartilage and normal-appearing subchondral bone. There was good integration of the implant with the surrounding tissue. Implantation of MSCs into cartilage defects appears to effect repair of both the articular cartilage and subchondral bone. Studies are ongoing to further characterize the use of MSCs for cartilage repair.

Auditory-verbal hallucinations and the phonological loop: a cognitive neuropsychological study.

A patient with continuous auditory-verbal hallucinations was studied, in comparison with two cases with a past history of similar hallucinations, from a cognitive neuropsychological perspective. This attempts to place hallucinations in the context of a normal cognitive process which has become disordered. The process in question is the phonological loop, equivalent to inner speech, derived from a model of short-term or working memory. A series of short-term memory tests, assumed to rely on the adequate functioning of the phonological loop, was administered, the results of which broadly conformed to a normal pattern of performance. It is concluded that verbal hallucinations cannot be regarded as involving the phonological loop directly. Other points in the short-term memory/language system at which verbal hallucinations could arise are discussed, as are suggestions for further research of this kind.

Pluripotent mesenchymal stem cells reside within avian connective tissue matrices.

Recent studies have noted the presence of putative stem cells derived from the connective tissues associated with skeletal muscle, heart, and dermis. Long-term continuous cultures of these cells from each tissue demonstrated five distinct phenotypes of mesodermal origin, i.e. muscle, fat, cartilage, bone, and connective tissue. Clonal analysis was performed to determine whether these morphologies were the result of a mixed population of lineage-committed stem cells or the differentiation of pluripotent stem cells or both. Putative stem cells from four tissues (skeletal muscle, dermis, atria, and ventricle) were isolated and cloned. Combined, 1158 clones were generated from the initial cloning and two subsequent subclonings. Plating efficiency approximated 5.8%. Approximately 70% of the 1158 clones displayed a pure stellate morphology, while the remaining clones contained a mixture of stellate, chondrogenic- or osteogenic-like morphologies or both. When cultured in the presence of dexamethasone, cells from all clones differentiated in a time- and concentration-dependent manner into muscle, fat, cartilage, and bone. These results suggest that pluripotent mesenchymal stem cells are present within the connective tissues of skeletal muscle, dermis, and heart and may prove useful for studies concerning the regulation of stem cell differentiation, wound healing, and tissue restoration, replacement and repair.

Epithelial abnormalities in intestine and kidney of the spontaneously hypertensive rat.

A variety of perturbations of calcium metabolism are reported to occur in the spontaneously hypertensive rat (SHR) compared to its genetic control the Wistar-Kyoto rat (WKY), including significant dysfunction of calcium handling by the proximal renal tubule of the SHR, resulting in impaired active calcium transport in the gut and an apparent renal calcium leak. We explored the intestinal and renal epithelia of 12- to 14-week-old SHR and WKY using electron microscopy. Biochemical comparisons of these transport epithelia included measurements of three vitamin D dependent cellular proteins and one structural protein: alkaline phosphatase, intestinal CaBP9K, renal CaBP28K, and villin expression. Electron microscopy demonstrated a patchy loss in microvilli in the SHR, accounting for approximately 10 to 15% of the total microvillar surface. In the kidney, morphological abnormalities were observed only in the proximal renal tubule. Again, there was patchy loss of microvilli from the brush border membrane. In SHR duodenal alkaline phosphatase activity was significantly reduced compared to the WKY (0.145 +/- 0.002 v 0.186 +/- 0.002 integrated extinction/min/micron 3 X 10(3) brush border (P less than .001). Duodenal CaBP9K and renal CaBP28K were significantly reduced in SHR compared to WKY. There were no differences in villin expression. These data are consistent with the previously characterized disturbances of active calcium transport in the intestine and inappropriate renal calcium leak in the SHR. While a possible link between these disturbances and hypertension remains to be determined, this study provides supportive evidence for a primary disturbance in cell calcium handling and transporting epithelia in this form of genetic hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)