Biomedical subjects

Prosper Benhaim

Publications and source records attributed to Prosper Benhaim.

At least 19 recordsLinked to original sources

PubMed2006

Monobloc advancement by distraction osteogenesis decreases morbidity and relapse.

BACKGROUND: Treatment of midface hypoplasia and forehead retrusion with monobloc advancement is associated with significant complications, including meningitis, prolonged intubation, and frontal bone flap necrosis. To see whether distraction of the monobloc segment offered decreased morbidity, the authors compared clinical outcomes of patients who underwent conventional monobloc advancement with those of patients who underwent monobloc distraction. METHODS: Group 1 (conventional monobloc; n = 12) underwent traditional monobloc advancement with bone grafting. Group 2 (modified monobloc; n = 11) did not receive ventriculoperitoneal shunts and underwent the above procedures with placement of a pericranial flap and fibrin glue over the midline defect. Group 3 (monobloc distraction; n = 24) underwent advancement of the monobloc segment by distraction osteogenesis using internal distraction devices. Complications included meningitis, cerebrospinal fluid leak, frontal bone flap loss, and wound infection. Preoperative, postoperative, and follow-up lateral cephalograms were used to assess horizontal changes of the forehead, midface, and maxilla. RESULTS: Group 3 (distraction monobloc) had the lowest complication rate (8 percent), followed by groups 2 (modified monobloc; 43 percent) and 1 (conventional monobloc; 61 percent) (p < 0.05). Group 3 achieved greater advancement (12.6 mm) than did group 2 (9.4 mm) or group 1 (9.1 mm) (p < 0.05). Relapse was least in group 3 (8 percent) compared with groups 2 (67 percent) and 1 (45 percent). CONCLUSIONS: Monobloc advancement by distraction osteogenesis had less morbidity and achieved greater advancement with less relapse compared with conventional methods of acute monobloc advancement with bone grafting. Monobloc distraction is superior to conventional methods of acute monobloc advancement and is an alternative to staged fronto-orbital advancement followed by Le Fort III advancement.

Adolescent↗

PubMed2006

Osteogenic potentiation of human adipose-derived stem cells in a 3-dimensional matrix.

Adipose-derived stem cells (ADSCs) hold promise for use in tissue engineering. Despite growing enthusiasm for use of ADSCs, there is limited research that has examined their behavior in different in vitro and in vivo systems. The purpose of our study was to evaluate the effect of the extracellular matrix structure and composition on osteogenic differentiation by comparing the osteogenic marker expression of ADSCs grown under 2-dimensional or 3-dimensional cell culture conditions. Group 1 (2-D) included ADSCs raised under conventional cell culture conditions (cells in a 2-D monolayer configuration) (n = 24), and group 2 (3-dimensional) included ADSCs seeded in a collagen gel (cells within a 3-dimensional, biologically active environment) (n = 24). Comparison of ADSC behavior between the 2 groups was analyzed during a 14-day time frame. Osteogenic marker expression (CBFA-1, alkaline phosphatase, osteonectin, osteopontin, Collagen I, and JNK2) was quantified by real-time PCR, and histologic analysis was performed. Histologically, group 1 (2-D) showed cell spreading and deposition of a calcified extracellular matrix. Group 2 (3-dimensional) assumed a disorganized state in the collagen gel, with extension of pseudopodia throughout the matrix. Expression of CBFA-1 was up-regulated immediately in both groups. However, cells in group 2 (3-dimensional) had a more rapid and greater overall expression compared with cells in group 1 (2-D) (250-fold greater at 4 days). At day 14, cells in group 2 (3-dimensional) showed greater expression of all other osteogenic markers than cells in group 1 (2-D) (2.3-fold greater expression of alkaline phosphatase [P < 0.05], 8.4-fold greater expression of osteonectin [P < 0.05], 6.4-fold greater expression of osteopontin [P < 0.05], 2.9-fold greater expression of collagen I [P < 0.05], and 2.5-fold greater expression of JNK2 [P < 0.05]). Our data showed there was a progressive stimulatory effect on ADSCs with regard to osteogenesis when cultured in a 3-dimensional gel compared with a 2-D monolayer.

Adipose Tissue↗

PubMed2005

Reconstruction of the interosseous membrane of the forearm with a graft substitute: a cadaveric study.

PURPOSE: Longitudinal radioulnar dissociation occurs when traumatic axial loading through the wrist disrupts the interosseous membrane (IOM) of the forearm and fractures the radial head (Essex-Lopresti injury). Proximal migration of the radius results in a wrist with a positive ulnar variance, which leads ultimately to painful ulnar-sided wrist degeneration and wrist pain during grasping activities that involve axial loading or ulnar deviation of the wrist. In theory reconstruction of the IOM with a graft substitute can limit proximal migration of the radius, thereby preserving wrist function. The objective of this study was to measure the abilities of 3 graft tissues to limit proximal radial displacement compared with the native IOM in a radial head-deficient cadaver model. METHODS: Sixteen fresh-frozen cadaveric forearms were loaded axially to 134 N through the potted central 3 metacarpals; the elbow was flexed to 90 degrees with the wrist in neutral rotation. Proximal displacement of the radius relative to the capitellum was measured. With the radial head excised specimens were first tested with the IOM intact. The IOM was then sectioned and central band IOM reconstructions were performed on each specimen using the following tissues: palmaris longus tendon, flexor carpi radialis (FCR) tendon, and a 1-cm- wide bone-patellar tendon-bone (BPTB) onlay allograft. Ten loading cycles were performed with each test configuration. Proximal radial displacement between 13.4 N and 134 N of applied wrist force was analyzed for the 10th loading cycle. The increase in proximal displacement between the first and 10th loading cycles (recorded at 134 N of wrist force) represented permanent elongation of the graft. RESULTS: Mean cross-sectional areas were 5.11 mm2 for the palmaris longus tendon, 15.23 mm2 for the FCR tendon, and 51.59 mm2 for the BPTB allograft. Mean proximal radial displacements were 3.04 mm (intact IOM), 4.37 mm (BPTB), 4.92 mm (FCR tendon), and 6.43 mm (palmaris tendon); all means were significantly different from each other. Mean permanent graft elongations were 0.06 mm (IOM), 0.36 mm (BPTB), 1.25 mm (FCR tendon), and 1.80 mm (palmaris tendon); all means were significantly different from each other with the exception of means for palmaris longus vs FCR and BPTB vs IOM. CONCLUSIONS: No graft reconstruction limited proximal radial displacement as effectively as the native IOM. Of the 3 graft tissues tested the BPTB allograft had the greatest cross-sectional area, allowed the least proximal radial displacement, and displayed the least permanent elongation after 10 cycles of loading. The relatively thin and narrow palmaris longus tendon appears to be the least desirable choice for IOM reconstruction because of its relatively low stiffness and tendency to elongate permanently after cyclic loading. When the radial head is absent rupture of the IOM allows unopposed proximal displacement of the radius relative to the ulna as the wrist is loaded axially. In the present tests all 3 graft tissues used to reconstruct the IOM limited proximal radial displacement. The choice of graft material is an important variable if IOM reconstruction is considered for treatment of an Essex-Lopresti injury.

Adult↗

PubMed2005

Graft reconstruction of the interosseous membrane in conjunction with metallic radial head replacement: a cadaveric study.

PURPOSE: Longitudinal radioulnar dissociation (Essex-Lopresti injury) occurs when traumatic axial loading through the wrist disrupts the interosseous membrane (IOM) of the forearm and fractures the radial head. Proximal migration of the radius results in an ulnar-positive wrist, which can lead to painful ulnar-sided wrist degeneration and distal radioulnar joint instability. The purpose of this study was to measure the ability of an IOM reconstruction used in combination with a metal prosthetic radial head implant to reduce distal ulnar forces in a cadaveric model. The effects of varying the initial graft pretension on distal ulnar force were also studied. METHODS: Twelve fresh frozen and thawed cadaveric forearms had a miniature load cell installed to record force in the distal ulna as the wrist was loaded axially to 134 N of compression force in neutral rotation. Intact forearms were tested first with the elbow in valgus and varus alignments. Loading tests were repeated after (1) insertion of a metal radial head implant that restored radius anatomic length, (2) excision of the IOM (with a radial head implant), and (3) reconstruction of the IOM using a palmaris longus tendon autograft (with a radial head implant). The implant then was removed and loading tests were repeated using 3 levels of initial graft pretension. RESULTS: Mean distal ulnar forces with an intact forearm were 23% of applied wrist force in the varus alignment and 12% in the valgus alignment. Mean force levels after insertion of the implant were 18% (varus) and 13% (valgus); these were not significantly different from corresponding values for the intact forearm. Mean force levels after section of the IOM were 30% (varus) and 14% (valgus); these were not significantly different from corresponding values for the intact forearm (varus and valgus) but the mean for varus was significantly greater than the corresponding value with an implant. After IOM reconstruction with a palmaris longus tendon tensioned to 22 N mean distal ulnar forces were 8% (varus) and 7% (valgus); these means were significantly less than the corresponding values for all prior test conditions. With the radial head removed increasing the level of graft pretension reduced significantly mean distal ulnar force. CONCLUSIONS: With the IOM resected insertion of a metal radial head implant alone did not reduce distal ulnar forces to intact forearm levels. When an IOM reconstruction was performed in combination with the implant mean distal ulnar force was reduced significantly to a level below that for the intact forearm. Applying pretension to the graft displaced the radius distally thereby making the wrist more ulnar negative and reducing distal ulnar force. Our results suggest that an IOM reconstruction used in combination with a metal radial head implant theoretically could help reduce distal ulnar impaction in an Essex-Lopresti injury.

Aged↗

PubMed2005

Effects of wafer resection and hemiresection from the distal ulna on load-sharing at the wrist: a cadaveric study.

PURPOSE: Ulnar impaction syndrome is commonly the result of a naturally occurring ulnar-positive condition, distal radius fracture malunion, or collapse of a fractured radial head. The Feldon wafer procedure and the Bowers distal hemiresection procedure are designed to decrease force transmitted through the distal ulna. The purpose of this study was to measure the effects of these procedures on distal ulnar loading with varying degrees of ulnar positivity at the wrist. METHODS: Using a specially designed miniature load cell distal ulnar force was measured in 20 fresh-frozen cadaveric forearms as the wrist was loaded axially to 134 N in neutral forearm rotation; tests were performed in valgus alignment with the elbow flexed to 90 degrees . Ulnar positivity (0 mm, +2 mm, +4 mm, and +6 mm) was created by incremental shortening of the distal radius using a sliding plate. The radial neck was sectioned transversely and the radial head fragment was fixed in its anatomic position using cemented metal prongs connected to a rigid bar; this allowed the radial head to be disconnected and tilted out of the loading pathway to simulate an excised radial head. Loading tests (with and without the radial head in place) were repeated after removal of a 3-mm wafer of bone beneath the triangular fibrocartilage complex, and again after a hemiresection of the distal ulna to the base of the ulnar styloid process. RESULTS: For each ulnar status condition (intact, wafer removal, hemiresection) the mean distal ulnar force generally increased as the wrist became more ulnar positive. Both wafer removal and hemiresection significantly decreased mean distal ulnar forces under all conditions of ulnar variance, with or without the radial head in place. With the radial head in place the mean distal ulnar forces (expressed as a percentage of applied wrist force) for the 0-mm condition were 16.9% (intact), 3.8% (wafer removal), and 3.5% (hemiresection); corresponding values for the +6-mm condition were 61.6% (intact), and 39.8% (wafer removal), 15.1% (hemiresection). With the radial head removed the mean distal ulnar forces for the 0-mm condition were 31.7% (intact), 4.6% (wafer removal), and 4.4% (hemiresection); corresponding values for the +6-mm condition were 96.4% (intact), 71.6%, (wafer removal), and 27.2% (hemiresection). The decrease of distal ulnar force resulting from hemiresection was significantly greater than that for wafer removal for all ulnar-positive conditions; force reductions were not significantly different between the 2 procedures with neutral ulnar variance. CONCLUSIONS: The results of the present study can help to offer a biomechanical basis for choosing between a Feldon wafer procedure and a Bowers hemiresection procedure in patients with ulnocarpal impaction syndrome. Both procedures produced equal decreases of distal ulnar force in the intact forearm. With an ulnar-positive wrist, the condition for which the procedure would be performed commonly, the hemiresection was more effective in decreasing force transmitted through the distal ulna.

Adult↗

PubMed2005

Tissue-engineered bone from BMP-2-transduced stem cells derived from human fat.

BACKGROUND: Progenitor cells capable of induction into multiple mesenchymal lineages have been isolated from human liposuction aspirates. These cells, named processed lipoaspirate cells, have previously shown in vitro osteogenic capacity. The purpose of this study was to examine the in vivo bone induction capacity of bone morphogenetic protein-2 (BMP-2)-transduced processed lipoaspirate cells using adipose tissue from multiple harvest sites. METHODS: Processed lipoaspirate cells extracted from human abdominal and buttock liposuction aspirates (n = 5) and from infrapatellar fat pads (n = 5) were placed in osteogenic media containing Dulbecco's Modified Eagle Medium with 10% fetal bovine serum supplemented with 50 muM ascorbic acid-2-phosphate and 10 mM beta-glycerol phosphate. Half of these cells were transfected with an adenovirus carrying the cDNA for bone morphogenetic protein-2 (adBMP-2). These transfected cells were then seeded onto collagen I matrices at a concentration of 2 x 10 cells/matrix and were placed into the hind limbs of severe combined immunodeficient mice (n = 10). Nontransfected processed lipoaspirate cells were placed in the contralateral limb as a control. After 6 weeks, specimens were analyzed by radiographs, densitometry, and hematoxylin and eosin and von Kossa staining. RESULTS: The average number of cells extracted from the abdominal/buttock lipoaspirates was 3.4 x 10 cells/100 ml fat aspirate and 5.5 x 10 cells per infrapatellar fat pad (average volume, 20.6 cc). All 10 BMP-2 transfected processed lipoaspirate constructs produced abundant radiographic and histologic bone. The bone was adequately mineralized and was beginning to establish a marrow cavity. There was no quantitative difference in bone production between harvest sites [mean, 2.0 +/- 0.1 aluminum units (knee) versus 2.1 +/- 0.1 aluminum units (abdomen/buttock); p = 0.14]. No bone was produced in the negative controls. CONCLUSIONS: Multipotential processed lipoaspirate cells can be extracted from adipose tissue harvested from liposuction aspirates or from the infrapatellar fat pad of the knee. Processed lipoaspirate cells can be transduced with the BMP-2 gene to produce abundant in vivo bone. These cells appear to be clinically useful for bone tissue engineering applications either as osteoprogenitor cells or as delivery vehicles for BMP-2.

Adenoviridae↗

PubMed2004

Bacteriostatic properties of biomatrices against common orthopaedic pathogens.

Tissue-engineered grafts for tissue regeneration include either mature or progenitor cells seeded onto biomatrices that provide shape and support for developing tissue. Popular biomaterials used in orthopaedic surgery include collagen type I, hyaluronic acid, hydroxyapatite, and polylactic polyglycolic acid (PLGA). Biomatrices with bacteriostatic properties may be beneficial in promoting tissue-engineered graft survival in patients susceptible to infection. We evaluated the bacteriostatic effects of these biomaterials on the growth of the four most common orthopaedic bacterial pathogens: Staphylococcus aureus, Staphylococcus epidermidis, beta-hemolytic Streptococcus, and Pseudomonas aeruginosa. Hyaluronic acid demonstrated the largest bacteriostatic effect on these pathogens by inhibiting bacterial growth by an average of 76.8% (p = 0.0005). Hydroxyapatite and collagen inhibited growth on average by 49.7% (p = 0.011) and 37.5% (p = 0.102), respectively. PLGA exhibited the least bacteriostasis with an average inhibition of 9.8% (NS) and actually accelerated the growth of beta-hemolytic Streptococcus and P. aeruginosa.

Anti-Bacterial Agents↗

PubMed2004

Chondrogenic potential of multipotential cells from human adipose tissue.

The use of stem cells for cell-based tissue-engineering strategies represents a promising alternative for the repair of cartilaginous defects. The multilineage potential of a population of putative mesodermal stem cells obtained from human lipoaspirates, termed processed lipoaspirate cells, was previously characterized. The chondrogenic potential of those cells was confirmed with a combination of histological and molecular approaches. Processed lipoaspirate cells under high-density micromass culture conditions, supplemented with transforming growth factor-beta1, insulin, transferrin, and ascorbic acid, formed well-defined nodules within 48 hours of induction and expressed the cartilaginous markers collagen type II, chondroitin-4-sulfate, and keratan sulfate. Reverse transcription polymerase chain reaction analysis confirmed the expression of collagen type II and the cartilage-specific proteoglycan aggrecan. In summary, human adipose tissue may represent a novel plentiful source of multipotential stem cells capable of undergoing chondrogenesis in vitro.

Adipose Tissue↗

PubMed2004

Load-sharing at the wrist following radial head replacement with a metal implant. A cadaveric study.

BACKGROUND: Surgical excision of the radial head is frequently required after a comminuted fracture of the radial head. The outcome of this procedure is often unpredictable, with some patients experiencing ulna-sided pain in the wrist secondary to proximal migration of the radius. Insertion of a radial head prosthesis could prevent proximal radial migration and restore normal load-sharing at the wrist. The thickness of the radial head implant is an important variable in restoring anatomical radial length; however, the effects of varying the length of implants that were used to reconstruct the radius on load-sharing at the wrist have not been studied biomechanically, to our knowledge. METHODS: A miniature load cell was attached to fifteen fresh-frozen cadaveric forearms to record force in the distal part of the ulna as the wrist was axially loaded to 134 N of compression force. Proximal displacement of the radius relative to the capitellum was also recorded. Loading tests on intact forearms were performed with the elbow in valgus and varus alignment and with three positions of wrist rotation (neutral, 45 degrees of pronation, and 45 degrees of supination). Loading tests were then repeated, with the same positions of varus and valgus elbow alignment and wrist rotation as had been used in the tests of the intact forearm, after radial head excision and subsequent insertion of metal radial head implants that restored anatomical length, implants that produced a radial length that was longer than the anatomical length, and implants that produced a radial length that was shorter than the anatomical length. Testing of these different implant thicknesses was repeated after sectioning of the interosseous membrane. RESULTS: The mean distal ulnar forces and mean proximal radial displacements following insertion of an implant that restored anatomical length were not significantly different from the corresponding values for the intact forearm. At neutral wrist rotation, replacing that implant with an implant that increased the radial length by 4 mm (after sectioning of the interosseous membrane) decreased the mean distal ulnar force from 13.4% to 3.3% of the applied wrist force with the elbow in valgus alignment and from 29.1% to 8.6% with the elbow in varus alignment. Replacing the implant that restored anatomical length with one that decreased the length by 4 mm (after sectioning of the interosseous membrane) significantly increased the mean distal ulnar force from 13.4% of the applied wrist load to 33.3% with the elbow in valgus alignment and from 29.1% to 51.6% with it in varus alignment. The mean distal ulnar forces were not significantly affected by the position of wrist rotation when the elbow was in valgus alignment. However, when the elbow was in varus alignment, the mean distal ulnar forces associated with all reconstructed radial lengths were significantly higher when the wrist was placed in 45 degrees of supination. CONCLUSIONS: In this cadaveric model, insertion of a metal implant maintained distal ulnar forces at normal levels, at all three positions of wrist rotation, when the radius had been restored to its original anatomical length. Distal ulnar forces and proximal radial displacements were significantly affected by the reconstructed length of the radius. CLINICAL RELEVANCE: Radial head implants are utilized to prevent proximal migration of the radius as the wrist is loaded; this is especially important when the interosseous membrane has been ruptured and thus cannot help to limit radial displacement. At the time of surgery, comminution and displacement of a radial head fracture may make estimation of the original radial length difficult. Our results demonstrate that, in terms of distal ulnar loading, it is preferable to insert an implant that is too thick rather than too thin.

Aged↗

PubMed2003

Differential expression of stem cell mobilization-associated molecules on multi-lineage cells from adipose tissue and bone marrow.

Our laboratory has characterized a population of stromal cells obtained from adipose tissue termed processed lipoaspirate cells (PLAs). PLAs, like bone-marrow derived mesenchymal stem cells (BM-MSCs), have the capacity to differentiate along the adipogenic, osteogenic, chondrogenic, and myogenic lineages, In order to better characterize these two multi-lineage populations, we examined the surface phenotype of both bone marrow and adipose tissue-derived cells from five patients undergoing surgery. PLA and BM-MSC cells were isolated, subcultivated, and evaluated for cell surface marker expression using flow cytometry. PLA and BM-MSC cells both expressed CD13, CD29, CD44, CD90, CD105, SH-3, and STRO-1. Differences in expression were noted for cell adhesion molecules CD49d (Integrin alpha4), CD54 (ICAM-1), CD34, and CD106 (VCAM-1). While markedly similar, the surface phenotypes of PLA and BM-MSC cells are distinct for several cell adhesion molecules implicated in hematopoietic stem cell homing, mobilization, and proliferation.

Adipose Tissue↗

PubMed2003

Multilineage cells from adipose tissue as gene delivery vehicles.

We have characterized a population of mesenchymal progenitor cells from adipose tissue, termed processed lipoaspirate (PLA) cells, which have multilineage potential similar to bone marrow-derived mesenchymal stem cells and are also easily expanded in culture. The primary benefit of using adipose tissue as a source of multilineage progenitor cells is its relative abundance and ease of procurement. We examined the infection of PLA cells with adenoviral, oncoretroviral, and lentiviral vectors. We demonstrate that PLA cells can be transduced with lentiviral vectors at high efficiency. PLA cells maintain transgene expression after differentiation into adipogenic and osteogenic lineages after lentiviral transduction. Therefore, PLA cells and lentiviral vectors may be an efficient combination for use as a therapeutic gene delivery vehicle.

Adenoviridae↗

PubMed2003

Bone induction by BMP-2 transduced stem cells derived from human fat.

PURPOSE: We have isolated pluripotent mesenchymal progenitor cells in large numbers from liposuction aspirates (processed lipoaspirate cells or PLAs). This study examines the osteogenic potential of PLAs and bone marrow aspirate cells (BMAs), when exposed to either recombinant human bone morphogenetic protein (BMP)-2 (rh-BMP-2) or adenovirus containing BMP-2 cDNA (Ad-BMP-2). METHODS: Liposuction aspirates underwent proteolytic digestion to obtain PLAs. After exposure to exogenous rh-BMP-2 or Ad-BMP-2 for four or seven days, PLAs and BMAs were assessed by histochemistry, spectrophotometry and RT-PCR. Western blotting and ELISA confirmed BMP gene transduction. Results were compared to osteoblasts and cells in osteogenic media only. PLA-Ad-BMP-2 cells were seeded on matrices and implanted in the hind limbs of SCID mice. RESULTS: Analysis of quantified bone precursor assays including extracellular ALP histomorphometry, intracellular ALP spectrophotometry, and calcified extracellular matrix (von Kossa) histomorphometry revealed that PLAs treated with exogenous rh-BMP-2 or transduced with a BMP-2 containing adenovirus (PLA-Ad-BMP-2) produced more bone precursors than osteoblasts (p=0.001). PLAs treated with exogenous rh-BMP-2 or PLA-Ad-BMP-2 also produced more bone precursors than BMAs (p=0.001), except for day 7 ALP histomorphometry (p=0.343). ELISA confirmed successful BMP-2 production by both progenitor cell groups transduced with Ad-BMP-2. H&E sections from collagen I matrices seeded with PLA-Ad-BMP-2 cells confirmed bone formation at six weeks. CONCLUSIONS: Liposuction aspirates contain PLAs that can be transfected with the BMP-2 gene, with rapid induction into the osteoblast phenotype at a rate comparable to rh-BMP-2 and osteoblast groups. Transduced PLAs produce more bone precursors with faster onset of calcified extracellular matrix than transduced BMAs. PLAs may be an ideal source of mesenchyme-lineage stem cells for gene therapy and tissue engineering.

Adenoviridae↗

PubMed2003

Decreased expression of fibroblast and keratinocyte growth factor isoforms and receptors during scarless repair.

Fibroblast growth factors (FGFs) are a family of 21 cytokines with a broad spectrum of activities, including regulation of cell proliferation, differentiation, and migration. The various FGFs bind to one or more of four different tyrosine kinase receptor types. FGFs 1, 2, 5, 7, and 10 are up-regulated during adult cutaneous wound healing. However, the expression of FGFs during fetal skin development and scarless wound healing has not been characterized. It was hypothesized that differential expression of FGF isoforms and receptors occurs during fetal skin development and that this differential expression pattern may regulate the transition from scarless repair to healing with scar formation. Excisional wounds (2 mm) were created on fetal rats at gestational days 16.5 (scarless) (one wound per fetus, n = 36 fetuses) and 19.5 (scarring) (one wound per fetus, n = 36 fetuses). Wounds were harvested at 24, 48, and 72 hours. Survival until wound harvest ranged from 66 to 75 percent for the gestational day 16 fetuses, and from 83 to 92 percent for the gestational day 19 fetuses. Nonwounded fetal skin from littermates (n = 12 fetuses per wound harvest time point) was used as the control. Wounds/skins were pooled by harvest time point, and RNA was isolated from pooled wounds/skins. Reduced-cycle, specific-primer reverse transcriptase-polymerase chain reaction was performed to determine the expression of FGF isoforms 2, 5, 7, 9, and 10 and FGF receptors 1, 2, and 4 in wounds relative to unwounded skin.In unwounded fetal skin, FGF isoform 5 expression more than doubled at birth. FGF 10 expression doubled during the transition period. FGF 7 expression increased more than sevenfold at birth. Expression of FGF isoforms 2 and 9 did not change during late fetal skin development. The expression of FGF receptors 1, 2, and 4 increased at birth. After wounding, expression of FGF isoforms 7 and 10 was down-regulated in scarless wounds, whereas FGF receptor 2 expression decreased in both scarless and scar-forming wounds. Expression of FGF isoforms 5 and 9 did not change in scarless wounds. FGF receptor 2 expression was down-regulated in both scarless and scarring wounds, but at an earlier and more sustained level in scarless wounds. Receptor type 4 expression increased in scarring wounds, whereas type 1 expression did not change in either scarless or scarring wounds. These results demonstrate an overall down-regulation of FGF expression during scarless healing.

Animals↗

PubMed2003

In vitro differentiation of human processed lipoaspirate cells into early neural progenitors.

Human processed lipoaspirate (PLA) cells are multipotent stem cells, capable of differentiating into multiple mesenchymal lineages (bone, cartilage, fat, and muscle). To date, differentiation to nonmesodermal fates has not been reported. This study demonstrates that PLA cells can be induced to differentiate into early neural progenitors, which are of an ectodermal origin. Undifferentiated cultures of human PLA cells expressed markers characteristic of neural cells such as neuron-specific enolase (NSE), vimentin, and neuron-specific nuclear protein (NeuN). After 2 weeks of treatment of PLA cells with isobutylmethylxanthine, indomethacin, and insulin, about 20 to 25 percent of the cells differentiated into cells with typical neural morphologic characteristics, accompanied by increased expression of NSE, vimentin, and the nerve-growth factor receptor trk-A. However, induced PLA cells did not express the mature neuronal marker, MAP, or the mature astrocyte marker, GFAP. It was also found that neurally induced PLA cells displayed a delayed-rectifier type K+ current (an early developmental ion channel) concomitantly with morphologic changes and increased expression of neural-specific markers. The authors concluded that human PLA cells might have the potential to differentiate in vitro into cells that represent early progenitors of neurons and/or glia.

Adult↗

PubMed2003

Comparison of multi-lineage cells from human adipose tissue and bone marrow.

Our laboratory has recently characterized a population of cells from adipose tissue, termed processed lipoaspirate (PLA) cells, which have multi-lineage potential similar to bone-marrow-derived mesenchymal stem cells (MSCs). This study is the first comparison of PLA cells and MSCs isolated from the same patient. No significant differences were observed for yield of adherent stromal cells, growth kinetics, cell senescence, multi-lineage differentiation capacity, and gene transduction efficiency. Adipose tissue is an abundant and easily procured source of PLA cells, which have a potential like MSCs for use in tissue-engineering applications and as gene delivery vehicles.

Adipose Tissue↗

PubMed2002

Human adipose tissue is a source of multipotent stem cells.

Much of the work conducted on adult stem cells has focused on mesenchymal stem cells (MSCs) found within the bone marrow stroma. Adipose tissue, like bone marrow, is derived from the embryonic mesenchyme and contains a stroma that is easily isolated. Preliminary studies have recently identified a putative stem cell population within the adipose stromal compartment. This cell population, termed processed lipoaspirate (PLA) cells, can be isolated from human lipoaspirates and, like MSCs, differentiate toward the osteogenic, adipogenic, myogenic, and chondrogenic lineages. To confirm whether adipose tissue contains stem cells, the PLA population and multiple clonal isolates were analyzed using several molecular and biochemical approaches. PLA cells expressed multiple CD marker antigens similar to those observed on MSCs. Mesodermal lineage induction of PLA cells and clones resulted in the expression of multiple lineage-specific genes and proteins. Furthermore, biochemical analysis also confirmed lineage-specific activity. In addition to mesodermal capacity, PLA cells and clones differentiated into putative neurogenic cells, exhibiting a neuronal-like morphology and expressing several proteins consistent with the neuronal phenotype. Finally, PLA cells exhibited unique characteristics distinct from those seen in MSCs, including differences in CD marker profile and gene expression.

Adipose Tissue↗

PubMed2002

Confocal microscopic analysis of scarless repair in the fetal rat: defining the transition.

Fetal wounds pass from scarless repair to healing with scar formation during gestation. This transition depends on both the size of the wound and the gestational age of the fetus. This study defines the transition period in the fetal rat model and provides new insight into scarless collagen wound architecture by using confocal microscopy. A total of 16 pregnant Sprague-Dawley rats were operated on. Open full-thickness wounds, 2 mm in diameter, were created on fetal rats at gestational ages 14.5 days (E14; n = 10), 16.5 days (E16; n = 42), and 18.5 days (E18; n = 42) (term = 21.5 days). Wounds were harvested at 24 (n = 18 per gestational age) and 72 hours (n = 24 per gestational age). Skin at identical gestational ages to wound harvest was used for controls. The wounds were fixed and stained with hematoxylin and eosin, antibody to type I collagen, and Sirius red for confocal microscopic evaluation. No E14 rat fetuses survived to wound harvest. Wounds created on E16 fetal rats healed completely and without scarring. E16 fetal rat hair follicle formation and collagen architecture was similar to that of normal, nonwounded skin. Wounds created on E18 fetal rats demonstrated slower healing; only 50 percent were completely healed at 72 hours compared with 100 percent of the E16 fetal rat wounds at 72 hours. Furthermore, the E18 wounds healed with collagen scar formation and without hair follicle formation. Confocal microscopy demonstrated that the collagen fibers were thin and arranged in a wispy pattern in E16 fetal rat wounds and in nonwounded dermis. E18 fetal rat wounds had thickened collagen fibers with large interfiber distances. Two-millimeter excisional E16 fetal rat wounds heal without scar formation and with regeneration of normal dermal and epidermal appendage architecture. E18 fetal rat wounds heal in a pattern similar to that of adult cutaneous wounds, with scar formation and absence of epidermal appendages. Confocal microscopy more clearly defined the dermal architecture in normal skin, scarless wounds, and scars. These data further define the transition period in the fetal rat wound model, which promises to be an effective system for the study of in vivo scarless wound healing.

Animals↗

PubMed2002

Myogenic differentiation by human processed lipoaspirate cells.

The use of undifferentiated cells for cell-based tissue engineering and regeneration strategies represents a promising approach for skeletal muscle repair. For such strategies to succeed, a readily available source of myogenic precursor cells must be identified. We have previously shown that cells isolated from raw human lipoaspirates, called processed lipoaspirate cells, display multilineage mesodermal potential in vitro. Because human liposuctioned fat is available in large quantities and can be harvested with low morbidity, it may be an ideal source of stem cells for tissue-engineering applications. In this study, processed lipoaspirate cells were isolated from raw lipoaspirates harvested from eight patients who underwent cosmetic surgery. Processed lipoaspirate cells were placed in promyogenic conditions for up to 6 weeks, and the expression of the myogenic markers MyoD1 and myosin heavy chain was confirmed by using structure, histology, and reverse transcriptase-polymerase chain reaction. Histologic results were quantitated as an indicator or myogenic differentiation levels. We found that induced human processed lipoaspirate cells form multinucleated cells after 3 weeks of induction, indicative of the formation of myotubes. In addition, MyoD1 and skeletal muscle myosin heavy chain are expressed at distinct time points during differentiation with MyoD1 expression preceding expression of myosin. Finally, approximately 15 percent of human processed lipoaspirate cells can be induced toward myogenic differentiation 6 weeks after induction. In summary, our findings suggest that human processed lipoaspirate cells differentiate into myogenic cells. Furthermore, these cells may be a useful source for skeletal muscle engineering and repair.

Adipocytes↗