Biomedical subjects

Lawrence J Bonassar

Publications and source records attributed to Lawrence J Bonassar.

At least 19 recordsLinked to original sources

PubMed2007

Binding and localization of recombinant lubricin to articular cartilage surfaces.

Lubricin is a secreted, cytoprotective glycoprotein that contributes to the essential boundary lubrication mechanisms necessary for maintaining low friction levels at articular cartilage surfaces. Diminishment of lubricin function is thereby implicated as an adverse contributing factor in degenerative joint diseases such as osteoarthritis. Lubricin occurs as a soluble component of synovial fluid, and is synthesized and localized in the superficial layer of articular cartilage (and thus has also been described as "superficial zone protein", or SZP); however, defined interactions responsible for lubricin retention at this site are not well characterized. In the current studies, we identified molecular determinants that enable lubricin to effectively bind to articular cartilage surfaces. Efficient and specific binding to the superficial zone was observed for synovial lubricin, as well as for recombinant full-length lubricin and a protein construct comprising the lubricin C-terminal (hemopexin-like) domain (LUB-C, encoded by exons 7-12). A construct representing the N-terminal region of lubricin (LUB-N, encoded by exons 2-5) exhibited no appreciable cartilage-binding ability, but displayed the capacity to dimerize, and thus potentially influence lubricin aggregation. Disulfide bond disruption significantly attenuated recombinant lubricin and LUB-C binding to cartilage surfaces, demonstrating a requirement for protein secondary structure in facilitating the appropriate localization of lubricin at relevant tissue interfaces. These findings help identify additional key attributes contributing to lubricin functionality, which would be expected to be instrumental in maintaining joint homeostasis.

Animals↗

PubMed2006

An allogenic cell-based implant for meniscal lesions.

BACKGROUND: Meniscal tears in the avascular zones do not heal. Although tissue-engineering approaches using cells seeded onto scaffolds could expand the indication for meniscal repair, harvesting autologous cells could cause additional trauma to the patient. Allogenic cells, however, could provide an unlimited amount of cells. HYPOTHESIS: Allogenic cells from 2 anatomical sources can repair lesions in the avascular region of the meniscus. STUDY DESIGN: Controlled laboratory study. METHODS: Both autologous and allogenic chondrocytes were seeded onto a Vicryl mesh scaffold and sutured into a bucket-handle lesion created in the medial menisci of 17 swine. Controls consisted of 3 swine knees treated with unseeded implants and controls from a previous experiment in which 4 swine were treated with suture only and 4 with no treatment. Menisci were harvested after 12 weeks and evaluated histologically for new tissue and percentage of interface healing surface; they were also evaluated statistically. RESULTS: The lesions were closed in 15 of 17 menisci. None of the control samples demonstrated healing. Histologic analysis of sequential cuts through the lesion showed formation of new scar-like tissue in all experimental samples. One of 8 menisci was completely healed in the allogenic group and 2 of 9 in the autologous group; the remaining samples were partially healed in both groups. No statistically significant differences in the percentage of healing were observed between the autologous and allogenic cell-based implants. CONCLUSION: Use of autologous and allogenic chondrocytes delivered via a biodegradable mesh enhanced healing of avascular meniscal lesions. CLINICAL RELEVANCE: This study demonstrates the potential of a tissue-engineered cellular repair of the meniscus using autologous and allogenic chondrocytes.

Absorbable Implants↗

PubMed2006

Review of injectable cartilage engineering using fibrin gel in mice and swine models.

More than a decade of work has been devoted to engineering cartilage for articular surface repair. This review covers the use of fibrin gel polymer as an injectable scaffold for generating new cartilage matrix from isolated articular chondrocytes beginning with studies in mice and culminating in an applied study in swine joints. These studies began with developing a formulation of fibrin that was injectable and promoted cartilage matrix formation. Subsequent studies addressed the problems of volume loss after the scaffolds were placed in vivo by adding lyophilized cartilage matrix. Additional studies focused on the ability of isolated chondrocytes to heal and repair cartilage in a model that could be biomechanically tested. In conclusion, this series of studies demonstrated that fibrin gel is a suitable polymer gel for generating new cartilage matrix from articular chondrocytes. The new matrix is capable of forming mechanical bonds between cartilage disks and can lead to healing and integration. Armed with these results, implantation of fibrin-cell constructs into defects in swine knees showed new cartilage formation and filling of the defects. Continuing work in these models with fibrin and other polymerizable hydrogels could result in a suitable cell-based therapy for articular cartilage lesions.

Animals↗

PubMed2006

Tissue-engineered lung: an in vivo and in vitro comparison of polyglycolic acid and pluronic F-127 hydrogel/somatic lung progenitor cell constructs to support tissue growth.

In this study, we describe the isolation and characterization of a population of adult-derived or somatic lung progenitor cells (SLPC) from adult mammalian lung tissue and the promotion of alveolar tissue growth by these cells (both in vitro and in vivo) after seeding onto synthetic polymer scaffolds. After extended in vitro culture, differentiating cells expressed Clara cell 10kDa protein, surfactant protein-C, and cytokeratin but did not form organized structures. When cells were combined with synthetic scaffolds, polyglycolic acid (PGA) or Pluronic F-127 (PF-127), and maintained in vitro or implanted in vivo, they expressed lung-specific markers for Clara cells, pneumocytes, and respiratory epithelium and organized into identifiable pulmonary structures (including those similar to alveoli and terminal bronchi), with evidence of smooth muscle development. Although PGA has been shown to be an excellent polymer for culture of specific cell types in vitro, in vivo culture in an immunocompetent host induced a foreign body response that altered the integrity of the developing lung tissue. Use of PF-127/cell constructs resulted in the development of tissue with less inflammatory reaction. These data suggest that the therapeutic use of engineered tissues requires both the use of specific cell phenotypes, as well as the careful selection of synthetic polymers, to facilitate the assembly of functional tissue.

Animals↗

PubMed2006

A novel approach to regenerating periodontal tissue by grafting autologous cultured periosteum.

In the field of oral and maxillofacial surgery, tissue-engineering techniques have been found useful in regenerating lost tissues. Periodontal disease causes severe destruction of periodontal tissue, including the alveolar bone. In this study we attempted to regenerate canine periodontal tissue defects by grafting autologous cultured membrane derived from the periosteum. Under appropriate culture conditions, periosteal cells produce enough extracellular matrix to form sheets. Periosteum specimens were peeled from the mandibular body of adult hybrid dogs and were cultured until cells formed membrane. ALP activity was measured to determine an optimal time for grafting. The cultured periosteum (CP) was grafted and sutured on a mechanically made Class III furcation defect in the 4th mandibular premolars. After 3 months, the samples were harvested and observed radiologically and histologically. In cases of CP, the bone defects were regenerated and filled with newly formed hard tissue, whereas in the controls the defects remained. These results show that our novel treatment is effective in regenerating alveolar bone for the treatment of periodontal disease.

Animals↗

PubMed2006

Direct freeform fabrication of seeded hydrogels in arbitrary geometries.

A major challenge in tissue engineering is the generation of cell-seeded implants with structures that mimic native tissue, both in anatomic geometries and intratissue cell distributions. By combining the strengths of injection molding tissue engineering with those of solid freeform fabrication (SFF), three-dimensional (3-D) pre-seeded implants were fabricated without custom-tooling, enabling efficient production of patient-specific implants. The incorporation of SFF technology also enabled the fabrication of geometrically complex, multiple-material implants with spatially heterogeneous properties that would otherwise be challenging to produce. Utilizing a custom-built robotic SFF platform and gel deposition tools, alginate hydrogel was used with calcium sulfate as a crosslinking agent to produce pre-seeded living implants of arbitrary geometries. The process was determined to be sterile and viable at 94 +/- 5%. The GAG and hydroxyproline production was found to be similar to that of other implants fabricated using the same materials with different shaping methods. The geometric fidelity of the process was quantified by using the printing platform as a computerized measurement machine (CMM); the RMS surface roughness of printed samples in the z-dimension was found to be 0.16 +/- 0.02 mm.

Alginates↗

PubMed2006

Tissue-engineered calcium alginate patches in the repair of chronic chinchilla tympanic membrane perforations.

OBJECTIVE: Chronic tympanic membrane perforations are a common problem in the United States. A high number of these cases results from placement of pressure equalization tubes. These perforations may initially be treated with paper patch techniques and although safe and well tolerated, the procedure demonstrates poor efficacy. The ideal treatment for small perforations should be rapid, minimally invasive, and efficacious. Calcium alginate-based tissue engineered tympanic membrane patches represent an attractive option, but in vivo data are required. METHODS: A controlled prospective study of tympanic membrane perforation repair using a well-known chinchilla model of chronic tympanic membrane perforation was performed. Calcium alginate-based tympanic membrane patches were created using computer-aided design techniques. A previously described chinchilla model of chronic tympanic membrane perforations was used to create stable perforations ranging from 2 to 5 mm. Ears with chronic perforations were divided into three groups: control (no patch), paper patch, and calcium alginate plugs. At 10 weeks postimplantation, all animals were killed and inspected both grossly and histologically for healing. RESULTS: In the chinchilla model, the alginate grafts demonstrated significantly improved healing rates over both the untreated control group (spontaneous repair) and the paper patch group; nine of 13 healed in the alginate group versus two of nine healed in the paper patch group (P < .05) versus one of 11 healed in the control group (P < .05). CONCLUSION: Calcium alginate tympanic membrane perforation patches offer a significant advantage in the repair of chronic perforations over traditional techniques in the chinchilla perforation model and may offer attractive opportunities in the clinical setting.

Alginates↗

PubMed2005

Matrix metalloproteinase activity synergizes with alpha2beta1 integrins to enhance collagen remodeling.

Cell-matrix interactions transmit a wealth of information about the extracellular environment. In return, a variety of responses from the cell are initiated by changes in the matrix. One such response involves the positive regulation of matrix metalloproteinases (MMPs) by alpha2beta1 integrin attaching to a specific extracellular matrix component, collagen. This study explores the relationship between mechanical and biochemical functions of alpha2beta1 integrins as it pertains to regulating matrix remodeling. To understand this relationship, the individual influences of MMP activity and alpha2beta1 integrin function on collagen gel contraction were studied. We have observed little evidence of mutual participation in matrix remodeling by the alpha2beta1 integrin and MMP activity in cell models where alpha2 is minimally expressed. In cells expressing high levels of alpha2, we see an increase in gel contraction that is enhanced by MMP activity. Measuring tension as it builds within the gel reveals that alpha2beta1 integrin presence correlates with force output but is insensitive to MMP activity. These data strongly suggest that alpha2beta1 regulates collagen gel remodeling through multiple simultaneous mechanisms including force generation and modulation of MMP activity.

Bone Remodeling↗

PubMed2005

A microfluidic biomaterial.

We report on the incorporation of microfluidic structure within a high-water-content hydrogel [4% (w/v) calcium alginate]. We used the microfluidic network to control the chemical environment within the hydrogel and demonstrated higher rates of delivery and extraction of solutes than was achievable by diffusion alone.

Alginates↗

PubMed2005

Biomechanical and biochemical characterization of composite tissue-engineered intervertebral discs.

Composite tissue-engineered intervertebral tissue was assembled in the shape of cylindrical disks composed of an outer shell of PGA mesh seeded with annulus fibrosus cells with an inner core of nucleus pulposus cells seeded into an alginate gel. Samples were implanted subcutaneously in athymic mice and retrieved at time points up to 16 weeks. At all retrieval times, samples maintained shape and contained regions of distinct tissue formation. Histology revealed progressive tissue formation with distinct morphological differences in tissue formation in regions seeded with annulus fibrosus and nucleus pulposus cells. Biochemical analysis indicated that DNA, proteoglycan, and collagen content in tissue-engineered discs increased with time, reaching >50% of the levels of native tissue by 16 weeks. The exception to this was the collagen content of the nucleus pulposus portion of the implants with were approximately 15% of native values. The equilibrium modulus of tissue-engineered discs was 49.0+/-13.2 kPa at 16 weeks, which was between the measured values for the modulus of annulus fibrosus and nucleus pulposus. The hydraulic permeability of tissue-engineered discs was 5.1+/-1.7x10(-14) m2/Pa at 16 weeks, which was between the measured values for the hydraulic permeability of annulus fibrosus and nucleus pulposus. These studies document the feasibility of creating composite tissue-engineered intevertebral disc implants with similar composition and mechanical properties to native tissue.

Alginates↗

PubMed2005

Poly(lactide-co-glycolide) microspheres as a moldable scaffold for cartilage tissue engineering.

This study demonstrates the use of biodegradable poly(lactide-co-glycolide) (PLG) microspheres as a moldable scaffold for cartilage tissue engineering. Chondrocytes were delivered to a cylindrical mold with or without PLG microspheres and cultured in vitro for up to 8 weeks. Cartilagenous tissue formed using chondrocytes and microspheres maintained thickness, shape, and chondrocyte collagen type II phenotype, as indicated by type II collagen staining. The presence of microspheres further enhanced total tissue mass and the amount of glycosaminoglycan that accumulated. Evaluation of microsphere composition demonstrated effects of polymer molecular weight, end group chemistry, and buffer inclusion on tissue-engineered cartilage growth. Higher molecular weight PLG resulted in a larger mass of cartilage-like tissue formed and a higher content of proteoglycans. Cartilage-like tissue formed using microspheres made from low molecular weight and free carboxylic acid end groups did not display increases in tissue mass, yet a modest increased proteoglycan accumulation was detected. Microspheres comprised of PLG with methyl ester end groups yielded a steady increase in tissue mass, with no real increase in matrix accumulation. The microencapsulation of Mg(OH)(2) had negative effects on tissue mass and matrix accumulation. The data herein reflect the potential utility of a moldable PLG-chondrocyte system for tissue-engineering applications.

Absorbable Implants↗

PubMed2004

Tissue-engineered composites of anulus fibrosus and nucleus pulposus for intervertebral disc replacement.

STUDY DESIGN: By the technique of tissue engineering, composite intervertebral disc implants were fabricated as novel materials for disc replacement, implanted into athymic mice, and removed at times up to 12 weeks. OBJECTIVES: The goal of this study was to construct composite intervertebral disc structures consisting of anulus fibrosus cells and nucleus pulposus cells seeded on polyglycolic acid and calcium alginate matrices, respectively. SUMMARY OF BACKGROUND DATA: Previous work has documented the growth of anulus fibrosus cells on collagen matrices and nucleus pulposus cells cultured on multiple matrices, but there is no documentation of composite disc implants. METHODS: Lumbar intervertebral discs were harvested from sheep spine, and the nucleus pulposus was separated from surrounding anulus fibrosus. Each tissue was digested in collagenase type II. After 3 weeks in culture, cells were seeded into implants. The shape of the anulus fibrosus scaffold was fabricated from polyglycolic acid and polylactic acid, and anulus fibrosus cells were pipetted onto the scaffold and allowed to attach for 1 day. Nucleus pulposus cells were suspended in 2% alginate and injected into the center of the anulus fibrosus. The disc implants were placed in the subcutaneous space of the dorsum of athymic mice and harvested at 4, 8, and 12 weeks. At each time point, 4 samples were stored in -70 degrees C for collagen typing and analysis of proteoglycan, hydroxyproline, and DNA. Other samples were fixed in 10% formalin for Safranin-O staining. RESULTS: The gross morphology and histology of engineered discs strongly resembled those of native intervertebral discs. Biochemical markers of matrix synthesis were present, increasing with time, and were similar to native tissue at 12 weeks. Tissue-engineered anulus fibrosus was rich in type I collagen but nucleus pulposus contained type II collagen, similar to the native disc. CONCLUSION: These results demonstrate the feasibility of creating a composite intervertebral disc with both anulusfibrosus and nucleus pulposus for clinical applications.

Animals↗

PubMed2004

Injectable tissue-engineered cartilage with different chondrocyte sources.

Injectable engineered cartilage that maintains a predictable shape and volume would allow recontouring of craniomaxillofacial irregularities with minimally invasive techniques. This study investigated how chondrocytes from different cartilage sources, encapsulated in fibrin polymer, affected construct mass and volume with time. Swine auricular, costal, and articular chondrocytes were isolated and mixed with fibrin polymer (cell concentration of 40 x 10 cells/ml for all groups). Eight samples (1 cm x 1 cm x 0.3 cm) per group were implanted into nude mice for each time period (4, 8, and 12 weeks). The dimensions and mass of each specimen were recorded before implantation and after explantation. Ratios comparing final measurements and original measurements were calculated. Histological, biochemical, and biomechanical analyses were performed. Histological evaluations (n = 3) indicated that new cartilaginous matrix was synthesized by the transplanted chondrocytes in all experimental groups. At 12 weeks, the ratios of dimension and mass (n = 8) for auricular chondrocyte constructs increased by 20 to 30 percent, the ratios for costal chondrocyte constructs were equal to the initial values, and the ratios for articular chondrocyte constructs decreased by 40 to 50 percent. Constructs made with auricular chondrocytes had the highest modulus (n = 3 to 5) and glycosaminoglycan content (n = 4 or 5) and the lowest permeability value (n = 3 to 5) and water content (n = 4 or 5). Constructs made with articular chondrocytes had the lowest modulus and glycosaminoglycan content and the highest permeability value and water content (p < 0.05). The amounts of hydroxyproline (n = 5) and DNA (n = 5) were not significantly different among the experimental groups (p > 0.05). It was possible to engineer injectable cartilage with chondrocytes from different sources, resulting in neocartilage with different properties. Although cartilage made with articular chondrocytes shrank and cartilage made with auricular chondrocytes overgrew, the injectable tissue-engineered cartilage made with costal chondrocytes was stable during the time periods studied. Furthermore, the biomechanical properties of the engineered cartilage made with auricular or costal chondrocytes were superior to those of cartilage made with articular chondrocytes, in this model.

Animals↗

PubMed2004

Analysis of bending behavior of native and engineered auricular and costal cartilage.

A large-deflection elasticity model was used to describe the mechanical behavior of cartilaginous tissues during three-point bending tests. Force-deflection curves were measured for 20-mm long x 4-mm wide x approximately 1-mm thick strips of porcine auricular and costal cartilage. Using a least-squares method with elastic modulus in bending as the only adjustable parameter, data were fit to a model based on the von Karman theory for large deflection of plates. This model described the data well, with an average RMS error of 14.8% and an average R(2) value of 0.98. Using this method, the bending modulus of auricular cartilage (4.6 MPa) was found to be statistically lower (p < 0.05) than that of costal cartilage (7.1 MPa). Material features of the cartilage samples influenced the mechanical behavior, including the orientation of the perichondrium in auricular cartilage. These methods also were used to determine the elastic moduli of engineered cartilage samples produced by seeding chondrocytes into fibrin glue. The modulus of tissue-engineered constructs increased statistically with time (p < 0.05), but still were statistically lower than the moduli of the native tissue samples (p > 0.05), reaching only about a third of the values of native samples.

Animals↗

PubMed2004

Effect of substrate mechanics on chondrocyte adhesion to modified alginate surfaces.

This study characterized the attachment of chondrocytes to RGD-functionalized alginate by examining the effect of substrate stiffness on cell attachment and morphology. Bovine chondrocytes were added to wells coated with 2% alginate or RGD-alginate. The alginate was crosslinked with divalent cations ranging from 1.25 to 62.5 mmol/g alginate. Attachment to RGD-alginate was 10-20 times higher than attachment to unmodified alginate and was significantly inhibited by antibodies to integrin subunits alpha3l and beta1, cytochalasin-D, and soluble RGD peptide. The equilibrium level and rate of attachment increased with crosslink density and substrate stiffness. Substrate stiffness also regulated chondrocyte morphology, which changed from a rounded shape with nebulous actin on weaker substrates to a predominantly flat morphology with actin stress fibers on stiffer substrates. The dependence of attachment on integrins and substrate stiffness suggests that chondrocyte integrins may play a role in sensing the mechanical properties of the matrices to which they are attached.

Alginates↗

PubMed2004

A novel injectable approach for cartilage formation in vivo using PLG microspheres.

This study documents the use of biodegradable poly(lactide-co-glycolide) (PLG) microspheres as a novel, injectable scaffold for cartilage tissue engineering. Chondrocytes were delivered via injection to the subcutaneous space of athymic mice in the presence and absence of PLG microspheres. Tissue formation was evaluated up to 8 weeks post-injection. Progressive cartilage formation was observed in samples containing microspheres. The presence of microspheres increased the quantity of tissue formed, the amount of glycosaminoglycan that accumulated, and the uniformity of type II collagen deposition. Microsphere composition influenced the growth of the tissue engineered cartilage. Higher molecular weight PLG resulted in a larger mass of cartilage formed and a higher content of proteoglycans. Microspheres comprised PLG with methyl ester end groups yielded increased tissue mass and matrix accumulation, but did not display homogenous matrix deposition. The microencapsulation of Mg(OH)2 had negative effects on tissue mass and matrix accumulation. Matrix accumulation, cell number, and tissue mass were unchanged by microsphere size, but larger microspheres increased the frequency of central necrosis in implants. The data herein reflect the promising utility of an injectable PLG-chondrocyte system for tissue engineering applications.

Animals↗

PubMed2004

A new histomorphometric method to assess growth plate chondrodysplasia and its application to the toothless (tl, Csf1(null)) osteopetrotic rat.

The proliferation and hypertrophy of growth plate chondrocytes set the pace and pattern for growth of endochondral bones. Complex signaling pathways regulating chondrocyte differentiation during development and growth have been discovered in recent years, but as yet little is known about how chondrocytes are able to orient themselves to align properly with respect to the direction of bone growth. Histomorphometric methods developed for analysis of growth plates rely to a significant extent on assessments of the relative heights of the zones of proliferating and hypertrophic chondrocytes. In a growing number of osteopetrotic mutations, however, it is becoming apparent that growth plates lack clearly demarcated zones of chondrocyte differentiation, and they show other notable histological abnormalities that cannot be measured with standard approaches. This is particularly true of mutations in which osteoclasts are altogether absent. We therefore developed a new approach that measures the salient features of this type of chondrodysplasia and have applied it to the toothless (tl) rat. The tl rat has a frameshift mutation in the Csf-1 gene that renders it null, resulting in severe osteopetrosis. An accompanying pathology is a severe, progressive growth plate chondrodysplasia. We measured cell orientation, cell area, and local columnar organization as functions of distance from the upper margin of the growth plate, in addition to growth plate thickness and cell density. All these parameters were markedly abnormal in the tl rats, thus implicating Csf-1 not only in its well-established role in regulating osteoclastic bone resorption, but also in endochondral ossification. This approach should prove useful in distinguishing among growth plate chondrodysplasias, most especially in the growing number of osteopetrotic mutations having growth plates that lack the normal zonal organization and in which the chondrocytes are mis-oriented. In turn, detailed assessments of chondrocyte misorientation may give insights into how they normally are able to arrange themselves with such precision.

Animals↗

PubMed2004

Fabrication of tissue engineered tympanic membrane patches using computer-aided design and injection molding.

OBJECTIVES/HYPOTHESIS: The goal of the current study was to use computer-aided design and injection molding technologies to tissue engineer precisely shaped cartilage in the shape of butterfly tympanic membrane patches out of chondrocyte-seeded calcium alginate gels. METHODS: Molds were designed on SolidWorks 2000 and built out of acrylonitrile butadiene styrene (ABS) using fused deposition modeling (FDM). Tympanic membrane patches were fabricated using bovine articular chondrocytes seeded at 50 x 10 cells/mL in 2% calcium alginate gels. Molded patches were cultured in vitro for up to 10 weeks and assessed biochemically, morphologically, and histologically. RESULTS: Unmolded patches demonstrated outstanding dimensional fidelity, with a volumetric precision of at least 3 microL, and maintained their shape well for up to 10 weeks of in vitro culture. Glycosaminoglycan and collagen content increased steadily over 10 weeks in culture, demonstrating continual deposition of new extracellular matrix consistent with new tissue development. CONCLUSIONS: The use of computer-aided design and injection molding technologies allows for the fabrication of very small, precisely shaped chondrocyte-seeded calcium alginate structures that faithfully maintain their shape during in vitro culture. In vitro fabrication of tympanic membrane patches with a precisely controlled geometry may have the potential to provide a minimally invasive alternative to traditional methods for the repair of chronic tympanic membrane perforations.

Analysis of Variance↗