Charles W. Patrick

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.

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.

Pore morphology effects on the fibrovascular tissue growth in porous polymer substrates.

The feasibility of developing biodegradable polymer scaffolds to engineer tissues was investigated by studying the effects of pore size on the dynamics of fibrovascular tissue ingrowth. Tissue advanced into amorphous poly(l-lactic acid) porous substrates faster as the pore diameter increased. Porous cylindrical devices of 13.5 mm diameter, 5 mm thickness, and approximately 500 μm pore size were filled completely by tissue 5 days postimplantation. Although prevascularized devices possessed minimal void volume for cell seeding to regenerate metabolic organs, they hold promise in the regeneration of tubular tissues by relying on the epithelization of prevascularized grafts.

Adipose tissue engineering: the future of breast and soft tissue reconstruction following tumor resection.

Reconstructive surgeons have always been at the forefront of medical technology. The history of reconstructive surgery began with ablative surgery, which was followed by tissue and organ transplantation, leading to contemporary tissue reconstruction. The field of reconstructive surgery is poised at the next stage of its evolution, namely tissue regeneration. The field of tissue engineering has largely defined this evolutionary leap. One active area of investigation is the development of tissue engineering strategies for adipose tissue. Bioengineers, life scientists, and reconstructive surgeons are synergistically coupling expertise in areas such as cell culture technology, tissue transfer, cell differentiation, angiogenesis, computer modeling, and polymer chemistry to regenerate adipose tissue de novo for breast replacement and soft-tissue augmentation following tumor resection. This work presents the current state of the art in adipose tissue engineering, as well the clinically translatable strategies currently under development. Semin. Surg. Oncol. 19:302-311, 2000.

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.

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.

Automated Selection of DAB-labeled Tissue for Immunohistochemical Quantification

The increased use of immunohistochemistry (IHC) in both clinical and basic research settings has led to the development of techniques for acquiring quantitative information from immunostains. Staining correlates with absolute protein levels and has been investigated as a clinical tool for patient diagnosis and prognosis. For these reasons, automated imaging methods have been developed in an attempt to standardize IHC analysis. We propose a novel imaging technique in which brightfield images of diaminobenzidene (DAB)-labeled antigens are converted to normalized blue images, allowing automated identification of positively stained tissue. A statistical analysis compared our method with seven previously published imaging techniques by measuring each ones agreement with manual analysis by two observers. Eighteen DAB-stained images showing a range of protein levels were used. Accuracy was assessed by calculating the percentage of pixels misclassified using each technique compared with a manual standard. Bland-Altman analysis was then used to show the extent to which misclassification affected staining quantification. Many of the techniques were inconsistent in classifying DAB staining due to background interference, but our method was statistically the most accurate and consistent across all staining levels.

Progress in adipose tissue construct development

Although the field of tissue engineering has been the focus of a great deal of promise and study, only recently has significant attention been given to the engineering of soft tissues. The applicability of an engineered adipose construct as a basic science model and a reconstructive tool is unquestioned; yet, there have been limitations in previous work, specifically issues of construct size and maintenance over time. This article briefly overviews the pivotal factors necessary for adipocyte growth and differentiation, optimal scaffolds for the engineering of soft tissues, and a means of providing vascular support for these highly demanding cells. Clinical science and bioengineering concepts that may provide the foundation toward the successful in vivo engineering of an adipose tissue construct that maintains its complex three-dimensional shape over time are critically reviewed.

The accuracy of stereolithography in planning craniofacial bone replacement.

Stereolithography can be used to produce physical models of the craniofacial skeleton from three-dimensional computed tomography (CT) data. The purpose of this study was to assess its accuracy for modeling osseous defects of the midface. Maxillary resections simulating unilateral maxillectomy (N = 3), bilateral maxillectomy (N = 3), and unilateral orbitomaxillectomy (N = 3) were performed as for sinus tumor resection on nine fresh cadaver skulls. Stereolithographic models (SLMs) were made from the specimens CT data. The accuracy of SLMs was determined by comparing distances between key landmarks on the skulls and SLMs. Each SLM was grossly accurate with some loss of thin delicate structures. The mean differences in overall dimensions between the SLMs and skull specimens were 1.5 mm (range: 0–5.5 mm) for craniofacial measures, 1.2 mm (range: 0–4.8 mm) for skull base measures, 1.6 (range: 0–5.8 mm) for midface measures, 1.9 mm (range: 0–7.9 mm) for maxilla measures, and 1.5 mm (range: 0–5.7 mm) for orbital measures. The mean differences in defect dimensions were 1.9 mm (range: 0.1–5.7 mm) for unilateral maxillectomy, 0.8 mm (range: 0.2–1.5 mm) for bilateral maxillectomy, and 2.5 mm (range: 0.2–7.0 mm) for orbitomaxillectomy defects. Midface SLMs may be more prone to error than those of other craniofacial regions because of the presence of thin walls and small projections. Thus, one should consider designing midface bone replacements that are larger in critical dimensions than those predicted by preoperative modeling. These findings have important implications for the planning of current surgical methods as well as future applications of tissue-engineered bone replacement.

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-κB consensus sites in fascin’s promoter and first intron, which appeared consistent with the greater endogenous transcriptional activities of TCF and NF-κB 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.