Biomedical subjects

C W Patrick

Publications and source records attributed to C W Patrick.

At least 19 recordsLinked to original sources

PubMed2002

Long-term implantation of preadipocyte-seeded PLGA scaffolds.

Studies were performed in a long-term effort to develop clinically translatable, tissue engineered adipose constructs for reconstructive, correctional, and cosmetic indications. Rat preadipocytes were harvested, isolated, expanded ex vivo, and seeded within PLGA scaffolds. Preadipocyte-seeded and acellular (control) scaffolds were implanted for 1-12 months. Explanted scaffolds were stained with osmium tetroxide, processed, and counterstained using H&E. Quantitative histomorphometric analysis was performed on all tissue sections to determine the amount of adipose tissue formed. Analyses revealed maximum adipose formation at 2 months, followed by a decrease at 3 months, and complete absence of adipose and PLGA at 5-12 months. These results extend a previous short-term study (Tissue Engineering 1999;5:134) and demonstrate that adipose tissue can be formed in vivo using tissue engineering strategies. However, the long-term maintenance of adipose tissue remains elusive.

Adipocytes↗

PubMed2001

Tissue engineering strategies for adipose tissue repair.

Tissue engineering is a relatively young field that combines engineering, clinical science, and life sciences to, in part, repair or regrow tissues. Adipose tissue has recently become a focus area for tissue engineering, encouraged by the large number of reconstructive, cosmetic, and correctional indications that could be addressed with clinically translatable adipose tissue engineering strategies. This review discusses the three aspects of an adipose construct, namely cell types, scaffold, and microenvironment, and presents current tissue engineering strategies under pursuit.

Adipose Tissue↗

PubMed2001

Muristerone A-induced nerve growth factor release from genetically engineered human dermal fibroblasts for peripheral nerve tissue engineering.

In this study, human dermal fibroblasts (hDFBs) were genetically modified to release human nerve growth factor (NGF) using an ecdysone-inducible system. NGF cDNA was inserted into the pIND vector and then hDFBs were cotransfected with pIND-NGF and pVgRXR. Muristerone A, an analog of ecdysone, was used as the inducing agent. NGF release from transfected hDFBs was assessed in vitro and in vivo. Transfected hDFBs in the presence of Muristerone A possessed a maximal in vitro release of 8.5 +/- 0.4 pg of NGF/mL per 10(3) cells, demonstrating significantly higher NGF levels compared to control hDFBs. The in vitro release rate curve for transfected hDFBs in the presence of Muristerone A exhibited a maximum of 5.1 +/- 0.2 ng NGF/10(6) cells/day. A PC-12 bioassay demonstrated that the in vitro NGF released is bioactive. When transfected hDFBs in the presence of Muristerone A were placed in vivo in nude rats, NGF levels reach 2074 +/- 257 pg/mL and 1620 +/- 132 pg/mL at 24 and 48 h, respectively. These levels were significantly higher than negative control and wound fluid levels. Results support further in vivo investigation of this molecular "on" switch for peripheral nerve regeneration.

Animals↗

PubMed2001

Dermal fibroblasts genetically engineered to release nerve growth factor.

Current surgical strategies for repair of critical nerves involves the transfer of normal donor nerve from an uninjured body location. One possible alternative to autogenous tissue replacement is the development of engineered constructs to replace those elements necessary for axonal proliferation. Delivery of growth factors is one strategy to enhance synthetic nerve constructs. Thus, this study focused on the delivery of nerve growth factor (NGF) by genetic engineering to begin approaching the microenvironment dictated, in part, by Schwann's cells. Rat dermal fibroblasts (DFBs) were modified genetically to release rat NGF. The reporter gene LacZ was used to assess the optimum nonviral transfection method commercially available before NGF transfection. FuGENE6 provided the optimum transfection efficiency (24% maximum, 20.1 +/- 1.9% 5-day average) as measured by beta-galactosidase catalytic activity. NGF release from transfected DFBs was assessed over a 3-day period. Compared with control (no transfection) DFBs and DFBs transfected with vector alone, DFBs transfected with an expression vector encoding rat beta-NGF demonstrated significantly (p < 0.05) higher levels of NGF, with a 3-day maximum of 111 pg NGF per milliliter. When normalized to cell number, NGF-transfected DFBs released 1.2 pg NGF per milliliter/10(3) cells. The NGF-transfected DFBs demonstrated a maximal NGF release rate at day 1 (1.2 ng NGF/10(6) cells per day), followed by a markedly lower, sustained release rate at days 2 and 3 (0.44 ng NGF/10(6) cells per day and 0.48 ng NGF/10(6) cells per day respectively). The release rate curves for control and vector-transfected DFBs also exhibited a maximal NGF release rate at day 1, but were followed by a decreasing release rate, potentially representing in vitro degradation of NGF present in fetal bovine serum. Although not first with the development of growth factor delivery through fibroblasts, these findings suggest that rat DFBs can be modified genetically to act like Schwann's cells to deliver NGF.

Animals↗

PubMed2000

C-erbB-2/ HER-2 upregulates fascin, an actin-bundling protein associated with cell motility, in human breast cancer cell lines.

The over-expression of c-erbB-2/ HER-2, a receptor tyrosine kinase, correlates with poor prognosis in patients with breast and ovarian cancer. In the human breast cancer cell line, MDA-MB-435, c-erbB-2 over-expression results in increased chemoinvasion and higher metastatic properties in nude mice. However, the mechanisms by which c-erbB-2 increases the malignant potential of cells remains unclear. We have determined that over-expression of c-erbB-2 in MDA-MB-435 cells, and in some additional breast cancer cell lines, is associated with graphic increases in mRNA and protein levels of the actin bundling protein fascin. Heightened fascin expression has been observed in other systems to result in greatly increased cell motility, and indeed, our work employing semi-automated time-lapse microscopy demonstrates that MDA-MB-435 cells over-expressing c-erbB-2 exhibit significantly heightened cellular dynamics and locomotion, while visualization of bundled microfilaments within fixed cells revealed enhanced formation of dendritic-like processes, microspikes and other dynamic actin based structures. To address the means by which c-erbB-2 over-expression might result in elevated fascin levels, we identified multiple perfect match TCF and NF-kappaB consensus sites in fascin's promoter and first intron, which appeared consistent with the greater endogenous transcriptional activities of TCF and NF-kappaB in c-erbB-2 over-expressing MDA-MB-435 cells. While such transcriptional modulation may occur in the context of the intact gene/chromatin, subsequent tests using reporter constructs did not support involvement of these signaling pathways. In conclusion, highly increased fascin levels were observed in MDA-MB-435 over-expressing c-erbB-2, likely contributing to these cells' altered actin dynamics, and increased cell motility and malignancy. Studies in progress aim to discern the means by which c-erbB-2 over-expression leads to transcriptional activation of the fascin gene.

Actins↗

PubMed2000

Development and in vitro characterization of vascular endothelial growth factor (VEGF)-loaded poly(DL-lactic-co-glycolic acid)/poly(ethylene glycol) microspheres using a solid encapsulation/single emulsion/solvent extraction technique.

Poly(DL-lactide-co-glycolide) (PLGA)/polyethylene glycol (PEG) microspheres are one modality of controlled delivery of biologically active molecules that would further the development of engineered tissues. As a possible mechanism to stimulate angiogenesis within an engineered tissue, vascular endothelial growth factor (VEGF) and bovine serum albumin (BSA) were coencapsulated into microspheres fabricated from PEG and 50/50 PLGA using a solid-encapsulation/single-emulsion/solvent extraction technique. Two VEGF/BSA ratios were studied: 1:2000 and 1:10,000. Analysis consisted of the loading efficiency, particle size distribution, bright-field microscopy, scanning electron microscopy, release kinetics, and an in vitro human umbilical vein endothelial cell proliferation assay to assess biological activity of the released VEGF. Results show the microspheres could be manufactured, stored, and degraded over 28 days. The burst release rates for 1:2000 and 1:10,000 VEGF/BSA microspheres were 71.87 +/- 8.11 and 27.91 +/- 1.71 ng/mL (mean +/- standard error of the mean), respectively; steady-state release rates were 6.56 +/- 1.10 and 2.21 +/- 0.47 ng/mL, respectively. The microspheres released biologically active VEGF, and the VEGF increased the proliferation of HUVECs in culture (p <.05). The successful development of a novel, cost-effective, scalable technique for producing microspheres loaded with biologically active proteins is presented. Using the data obtained from these studies, a defined concentration of microspheres will deliver a quantifiable level of VEGF at a known release rate.

Animals↗

PubMed2000

Clinical long-term in vivo evaluation of poly(L-lactic acid) porous conduits for peripheral nerve regeneration.

It was the purpose of this study to evaluate the clinical long-term effects of PLLA degradation in vivo on nerve regeneration in the rat sciatic nerve model. Thirty-one Sprague Dawley rats were utilized. Two groups of animals were selected. The control group of 10 animals received a 12 mm reversed isograft into the right sciatic nerve from 5 donor animals. The experimental group (n = 21) received a 12 mm empty PLLA conduits placed into a 12 mm defect in the right sciatic nerve. The left leg served as an internal control. Walking track analysis was performed monthly through 8 months. At the end of 4 and 8 months, animals in the control isograft and experimental group had the medial and lateral gastrocnemius muscles harvested and weighed for comparison. The midconduit/isograft and the distal nerve in these same animals were harvested and histomorphologically analyzed. Multiple samples were collected and expressed as means +/- standard error. A two-sample t-test and Wilcoxon rank sum test was used to compare the variables. Significance level was set at alpha = 0.05. After Bonferroni correction for multiple testing, a p value of < or = 0.01 was considered statistically significant. Throughout all time periods, the PLLA conduit remained structurally intact and demonstrated tissue incorporation and vascularization. There was no evidence of conduit collapse or breakage with limb ambulation. Moreover, there was no evidence of conduit elongation at 8 months as previously observed with the 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) conduits. The mean absolute value of the sciatic functional index (SFI) demonstrated no group differences from isograft controls measured over the 8 months except at 3 months where the isograft values were higher (p = 0.0379) and at 7 months were the isograft group was significantly lower (p = 0.0115). At 4 and 8 months, the weight of the gastrocnemius muscles of the experimental group was not significantly different from isografts. At 4 months the number of axons/mm2 and nerve fiber density was not significantly different between the isograft control and experimental groups in either the midconduit/isograft or distal nerve. At 8 months the number of axons/mm2 was significantly lower in the isograft compared to the midconduit experimental group (p = 0.006). The number of axons/mm2 in the distal nerve and the nerve fiber density in the midconduit and distal nerve were not significantly different between the two groups. The study confirmed our initial hypothesis that PLLA conduits are a viable scaffold for clinical long-term nerve gap replacement. We are critically aware however that longer evaluation of polymer degradation is warrented. Further studies on these individual nerve components are continuing, with the ultimate goal being the fabrication of a bioactive conduit that meets or exceeds the functional results of isografts.

Animals↗

PubMed1999

Measurement of blood flow and oxygen tension in adjacent tissues in pedicled and free flap head and neck reconstruction.

The purpose of this study was to evaluate blood flow and transcutaneous partial oxygen pressure (TcPO(2)) in adjacent tissues to free and pedicled flaps following reconstructive procedures used in conjunction with radical surgery for head and neck cancers. Fifty patients were included. Fourteen patients had reconstruction with pedicled flaps and 36 with free flaps. For each patient, TcPO(2) and laser Doppler flow measurements were taken at the center of the flap, in adjacent tissue, and in a corresponding contralateral site. Three laser Doppler measurements were performed at each site and a mean value recorded. All patients had undergone reconstruction up to 6 months prior to the time of the measurements. The collected data were analyzed using a Wilcoxon signed rank test. There were no statistically significant differences in partial oxygen tension or laser Doppler values between tissues adjacent to free compared to pedicled flaps. Although there is strong evidence to support that free flaps have improved blood flow and partial oxygen tension over pedicled flaps, further study is required to evaluate adjacent tissues. Flap choice may assist with alteration in blood flow in less favorable defects such as those in previously irradiated fields and those resulting from burn scars or chronic infections.

Adolescent↗

PubMed1999

In vivo evaluation of poly(L-lactic acid) porous conduits for peripheral nerve regeneration.

The present study provides in vivo trials of poly(L-lactic acid) (PLLA) as a porous biodegradable nerve conduit using a 10 mm sciatic nerve defect model in rats. The PLLA conduits, fabricated by an extrusion technique, had an inner diameter of 1.6 mm, an outer diameter of 3.2 mm, and a length of 12 mm. They were highly porous with an interconnected pore structure (of 83.5% porosity and 12.1 microm mean pore size). The conduits were interposed into the right sciatic nerve defect of Sprague Dawley rats using microsurgical techniques; nerve isografts served as controls. Walking track analysis was performed after conduit placement monthly through 16 weeks. At the conclusion of 6 and 16 weeks, sections from the isograft/conduit and distal nerve were harvested for histomorphometric analysis. The right gastrocnemius muscle was also harvested and its weight was determined. All conduits remained intact without breakage. Moreover, no conduit elongated during the 16 weeks of placement. Walking track analysis and gastrocnemius muscle weight demonstrated increasing regeneration over the 16 weeks in both the conduit and isograft control groups, with control values significantly greater. The nerve fiber density in the distal sciatic nerve for the PLLA conduits (0.16+/-0.07) was similar to that for the control isografts (0.19+/-0.05) at 16 weeks. The number of axons/mm2 in the distal sciatic nerve for the PLLA conduits was lower than that for the isografts (13 800+/-2500 vs. 10700+/-4700) at 16 weeks. The results for PLLA were significantly improved over those for 75:25 poly(DL-lactic-co-glycolic acid) of a previous study and suggest that PLLA porous conduits may serve as a scaffold for peripheral nerve regeneration.

Animals↗

PubMed1999

Preadipocyte seeded PLGA scaffolds for adipose tissue engineering.

Adipose tissue equivalents have not been addressed as yet despite the clinical need in congenital deformities, posttraumatic repair, cancer rehabilitation, and other soft tissue defects. Preadipocytes were successfully harvested from rat epididymal fat pads of Sprague-Dawley and Lewis rats and expanded ex vivo. In vitro cultures demonstrated full differentiation of preadipocytes into mature adipocytes with normal lipogenic activity. The onset of differentiation was well-controlled by regulating preadipocyte confluency. Poly(lactic-co-glycolic) acid (PLGA) polymer disks with 90% porosity, 2.5 mm thick, 12 mm diameter, pore size range of 135-633 microm were fabricated and seeded with preadipocytes at 10(5) cells/mL. Disks in vitro demonstrated fully differentiated mature adipocytes within the pores of the disks. Short-term in vivo experiments were conducted by implanting preseeded disks subcutaneously on the flanks of rats for 2 and 5 weeks. Histologic staining of harvested disks with osmium tetroxide (OsO4) revealed the formation of adipose tissue throughout the disks. Fluorescence labeling of preadipocytes confirmed that formed adipose tissue originated from seeded preadipocytes rather than from possible infiltrating perivascular tissue. This study demonstrates the potential of using primary preadipocytes as a cell source in cell-seeded polymer scaffolds for tissue engineering applications.

Adipocytes↗

PubMed1999

The effects of cisplatinum and vincristine on peripheral nerve regeneration.

Current treatment modalities for extremity sarcoma often include tumor extirpation plus neoadjuvant therapy. Limb-sparing surgery may require reconstruction of critical nerve defects. Neurotoxic side effects from adjuvant chemotherapy have been reported and raise concerns regarding the effects of chemotherapy on nerve regeneration. In an attempt to define the effects of adjuvant chemotherapy on peripheral nerve regeneration, cisplatin and vincristine were administered to rats following isografting of the posterior tibial nerve. Parameters used to assess peripheral nerve regeneration included walking track analysis and histomorphology. Sixty 250-g Sprague-Dawley rats were randomly allocated into one of three treatment groups. Each animal underwent a 15-mm reversed interposition nerve isograft from 30 donor rats into the right posterior tibial nerve. Ten animals served as control. The remaining animals were divided into two groups of 25 animals each. One group received cisplatin (75 mg/m2) and the other group received vincristine (1 mg/m2). Chemotherapy was administered at 4-week cycles for a total of six cycles (24 weeks). Walking track analysis was performed monthly. Nerve specimens were harvested from the grafted segment and the distal posterior tibial nerve for histomorphology. Walking track analysis demonstrated no statistical difference in print length between the control and chemotherapeutic groups at the conclusion of the study. The number of axons per square millimeter and nerve fiber density were not statistically different between control and chemotherapeutic groups. In the rodent posterior tibial nerve model, postoperative adjuvant therapy does not significantly alter functional outcome in peripheral nerve regeneration. The practice of immediate nerve grafting after tumor extirpation, despite planned postoperative chemotherapy, is supported.

Animals↗

PubMed1998

Manufacture of porous biodegradable polymer conduits by an extrusion process for guided tissue regeneration.

We have fabricated porous, biodegradable tubular conduits for guided tissue regeneration using a combined solvent casting and extrusion technique. The biodegradable polymers used in this study were poly(DL-lactic-co-glycolic acid) (PLGA) and poly(L-lactic acid) (PLLA). A polymer/salt composite was first prepared by a solvent casting process. After drying, the composite was extruded to form a tubular construct. The salt particles in the construct were then leached out leaving a conduit with an open-pore structure. PLGA was studied as a model polymer to analyze the effects of salt weight fraction, salt particle size, and processing temperature on porosity and pore size of the extruded conduits. The porosity and pore size were found to increase with increasing salt weight fraction. Increasing the salt particle size increased the pore diameter but did not affect the porosity. High extrusion temperatures decreased the pore diameter without altering the porosity. Greater decrease in molecular weight was observed for conduits manufactured at higher temperatures. The mechanical properties of both PLGA and PLLA conduits were tested after degradation in vitro for up to 8 weeks. The modulus and failure strength of PLLA conduits were approximately 10 times higher than those of PLGA conduits. Failure strain was similar for both conduits. After degradation for 8 weeks, the molecular weights of the PLGA and PLLA conduits decreased to 38% and 43% of the initial values, respectively. However, both conduits maintained their shape and did not collapse. The PLGA also remained amorphous throughout the time course, while the crystallinity of PLLA increased from 5.2% to 11.5%. The potential of seeding the conduits with cells for transplantation or with biodegradable polymer microparticles for drug delivery was also tested with dyed microspheres. These porous tubular structures hold great promise for the regeneration of tissues which require tubular scaffolds such as peripheral nerve, long bone, intestine, or blood vessel.

Biocompatible Materials↗

PubMed1998

The effects of preoperative irradiation on peripheral nerve regeneration.

The purpose of this study was to evaluate the effects of preoperative external cobalt60 beam irradiation on nerve regeneration. Ninety 250-g male Sprague-Dawley rats were studied. Peripheral nerve regeneration was measured by walking track analysis and histomorphology of the proximal, grafted, and distal nerve segments. Ninety animals were randomly assigned to one of five treatment groups, receiving a total fractionated dose of 30, 50, 70, and 90 Gy. Each animal received a 15-mm interposition nerve graft into the right posterior tibial nerve 6 weeks following completion of radiation therapy. The left leg served as a control. The remaining 10 animals received a nerve isograft subjected to a single dose of 30 Gy prior to placement (group 5). Walking track analysis was performed monthly through 8 months. At the conclusion of 120 and 240 days, sections of the proximal, grafted, and distal nerve were harvested, stained, and examined histomorphologically. Evaluation of the print length index demonstrated no statistical difference between our previously established nonirradiated controls, the irradiated groups, and the irradiated isograft group (group 5). The total number of axons per square millimeter was significantly decreased in the distal segment of all irradiated groups when compared with the controls. No statistical difference in number of axons per square millimeter was noted in the irradiated isograft group. Furthermore, no statistical difference was noted in the nerve fiber density between the control group, the preoperative irradiated groups, or the irradiated isograft group (group 5). Despite the reduction in myelinated regenerating fibers, no reduction in function was observed as measured by walking track analysis. Thus, immediate reconstruction of peripheral nerve defects in the face of preoperative irradiation may not be contraindicated.

Animals↗

PubMed1998

Computer-assisted histometric analysis of tissue-engineered ovine bone.

OBJECTIVE: To develop a computer-assisted histometric technique that quantitatively determines the amount of regenerating bone, while excluding fibrovascular tissue and void spaces, in tissue-engineered bone constructs. To this end, a histometric technique was developed that couples digital tiling with adaptive, multiband color thresholding (AMBCT). STUDY DESIGN: To test the technique, a previously described model bone tissue-engineered construct filled with morcellized bone graft was employed. Histometric techniques were applied to quantify the amount of bone formed following eight weeks of implantation. RESULTS: The histometric technique was able to yield quantitative information regarding the amount of bone despite intrahistologic and interhistologic differences in staining. The technique is user friendly and highly automated. In addition to area fractions, the technique can provide bone ingrowth profiles as a function of geometry and implantation time. CONCLUSION: Digital tiling coupled with AMBCT offers an easy, fast and reproducible technique that aids in quantification of bone within histologic sections. In addition, the technique can be adapted to quantification of other tissues. Further studies are under way to investigate the potential of correlating the histometric technique with mechanical strength analyses of tissue-engineered bone specimens.

Animals↗

PubMed1997

Intracellular pH changes in human aortic smooth muscle cells in response to fluid shear stress.

The smooth muscle cell (SMC) layers of human arteries may be exposed to blood flow after endothelium denudation, for example, following balloon angioplasty treatment. These SMCs are also constantly subjected to pressure driven transmural fluid flow. Flow-induced shear stress can alter SMC growth and metabolism. Signal transduction mechanisms involved in these flow effects on SMCs are still poorly understood. In this work, the hypothesis that shear stress alters the intracellular pH (pHi) of SMC is examined. When exposed to venous and arterial levels of shear stress, human aortic smooth muscle cells (hASMC) undergo alkalinization. The alkalinization plateau persisted even after 20 min of cell exposure to flow. Addition of amiloride (10 micromoles) or its 5-(N-ethyl-N-isopropyl) analog (EIPA, 10 micromoles), both Na+/H+ exchanger inhibitors, attenuated intracellular alkalinization, suggesting the involvement of the Na+/H+ exchanger in this response. The same concentrations of these inhibitors did not show an effect on pHi of hASMCs in static culture. 4-Acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid (SITS, 1 mM), a Cl-/HCO3- exchange inhibitor, affected the pHi of hASMCs both in static and flow conditions. Our results suggest that flow may perturb the Na+/H+ exchanger leading to an alkalinization of hASMCs, a different response from the flow-induced acidification seen with endothelial cells at the same levels of shear stress. Understanding the flow-induced signal transduction pathways in the vascular cells is of great importance in the tissue engineering of vascular grafts. In the case of SMCs, the involvement of pHi changes in nitric oxide production and proliferation regulation highlights further the significance of such studies.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1996

Quantitative effects of peripheral monocytes and nerve growth factor on CNS neural morphometric outgrowth parameters in vitro.

Would healing of the central nervous system (CNS) is a complex process involving interactions between cells from both the vascular and the neural environments, extracellular matrix proteins, and a cocktail of agonistic and antagonistic bioactive molecules. Vascular cells, particularly peripheral monocytes and macrophages, are believed to play an important role in organizing and mediating CNS tissue reactions subsequent to penetrating injuries that compromise the blood-brain barrier. Although many investigators have studied the effect of macrophages and microglia (resident brain macrophages) on neural outgrowth, little is known regarding monocyte effects. We have combined tissue culture, video microscopy, and digital image processing and analysis to quantify morphometric parameters of neurons exposed to monocyte secretory products in vitro. The experimental system developed is simple in design but provides a quantitative understanding of cellular function and molecular mechanisms and has the ability to both study processes of graded complexity and relate cellular function to overall systems behavior. We evaluate the efficacy of the experimental model developed by measuring morphometric parameters of human neural cells (hNT cell line) in the presence of nerve growth factor (NGF). Results suggest that monocyte-conditioned media (MCM) increases neuron outgrowth parameters, such as neuritic output, mean arbor output, neurite branching, and effective cell diameter. Moreover, we show that the bioactive factor present in MCM is not IL-1 and the activity of the factor with respect to neural outgrowth is between that of 10 and 100 ng/ml NGF.

Cell Line↗

PubMed1996

Cellular interactions among marrow stromal and normal/neoplastic pre-B- and B-lymphoblastic cells.

The direct interaction between B-cells, both normal and neoplastic, and marrow stromal cells (MSC) or MSC-secreted extracellular matrix proteins is believed to play a decisive role in lymphopoiesis as well as in the sustained proliferation and homing of lymphoid malignancies. Although information has been accruing on possible obligatory cell-cell and cell-extracellular matrix adhesion molecules, our current state of knowledge regarding B-cell interaction with the marrow microenvironment is rather limited. We discuss in this review what is currently known regarding adhesion molecules involved in the apposition between B-cells and MSC. In addition, we describe how the cellular interactions are modulated by cytokines present in the marrow microenvironment. Further, we present possible signal transduction pathways activated by B-cell/MSC apposition and attempt to integrate in vitro data with in vivo observations. We close this review by providing implications for novel clinical and therapeutic applications based on the current knowledge of B-cell/MSC adhesion mechanisms and signaling pathways.

B-Lymphocytes↗

PubMed1995

Heterotypic adherence between human B-lymphoblastic and pre-B-lymphoblastic cells and marrow stromal cells is a biphasic event: integrin very late antigen-4 alpha mediates only the early phase of the heterotypic adhesion.

Heterotypic adherence between marrow stromal cells (MSC) and lymphoblastic cells is essential for normal lymphopoiesis and malignant lymphoblastic development. However, the detailed molecular mechanisms by which this heterotypic adherence occurs are poorly understood. The cell-cell interactions between a B-lymphoblastic cell line (UTMB-460) and a pre-B-cell line (NALM-6) with MSC were chosen as models to investigate potential mechanisms and adhesion molecules involved in the apposition between normal and malignant lymphoblastic cells and MSC. A parallel-flow detachment assay (PFDA) and a 51Cr detachment assay, coupled with monoclonal antibody (MoAb) blocking experiments, were used to quantify the attachment of lymphoblastic cells to confluent monolayers of MSC. The apposition between MSC and B-lymphoblastic cells (UTMB-460 cells) was investigated for variable time periods, ranging from 1 minute to 4 hours. Results from the temporal study suggest that the heterotypic adherence of the B-lymphoblastic cells to MSC is a biphasic event and the interactions occur rapidly (< or = 1 minute) after the two cells come into contact. More specifically, the early phase of adherence (< or = 15 minutes) solely involves very late antigen-4 alpha (VLA-4 alpha)/vascular cell adhesion molecule 1 (VCAM-1) interactions, as evidenced by the nearly complete inhibition (93%) of UTMB-460 cell adherence in the presence of anti-VLA-4 alpha. The late phase (> or = 30 minutes) proceeds despite the continuous presence of anti-VLA-4 alpha. In addition, the late-phase adherence is not affected by MoAbs to LFA-1, CD44, VCAM-1, E-selectin, or L-selectin, which suggests the possible involvement of other adhesion molecules. Adherence of pre-B-lymphoblastic cells (NALM-6) to MSC is also biphasic. Integrin VLA-4 is again a major player in the early phase of pre-B-lymphoblastic cell/MSC interactions. The early phase of adherence may be important in homing of the malignant lymphoblastic cells to the MSC and the late phase in retention of malignant lymphoblastic cells in the bone marrow.

Antibodies, Monoclonal↗