Expression of superficial zone protein in mandibular condyle cartilage.

OBJECTIVE: Superficial zone protein (SZP) has been shown to function in the boundary lubrication of articular cartilages of the extremities. However, the expression of SZP has not been clarified in mandibular cartilage which is a tissue that includes a thick fibrous layer on the surface. This study was conducted to clarify the distribution of SZP on the mandibular condyle and the regulatory effects of humoral factors on the expression in both explants and fibroblasts derived from mandibular condyle. METHODS: The distribution of SZP was determined in bovine mandibular condyle cartilage, and the effects of interleukin-1beta (IL-1beta) and transforming growth factor-beta (TGF-beta) on SZP expression were examined in condyle explants and fibroblasts derived from the fibrous zone of condyle cartilage. RESULTS: SZP was highly distributed in the superficial zone of intact condyle cartilage. The SZP expression was up-regulated by TGF-beta in both explants and cultured fibroblasts, whereas the expression was slightly down-regulated by IL-1beta. A significant increase in accumulation of SZP protein was also observed in the culture medium of the fibroblasts treated with TGF-beta. CONCLUSIONS: These results suggest that SZP plays an important role in boundary lubrication of mandible condylar cartilage, is synthesized locally within the condyle itself, and exhibits differential regulation by cell mediators relevant to mandibular condyle repairing and pathologies.

Microscale three-dimensional polymeric platforms for in vitro cell culture systems.

This paper describes fabrication schemes to create multidimensional polymeric platforms to study cell function. A key feature of these constructs is the replication of in vivo geometry and dimensional size scales that will aid in the understanding of fundamental cell-environment interactions. Advantages of these microtextured membranes include the high degree of reproducibility, optical clarity, and the ability to create multiple features on the micron and sub-micron size scale. We have demonstrated the creation of controlled microscale features on hydrogels as well as biodegradable materials such as poly(lactic-glycolic acid). These microtopographies selectively degrade under physiological conditions. Because of the flexibility of substrate material and the ease of creating micron size structures, this technique can be applied to a multitude of physiological and biological systems.

Nanoporous biocapsules for the encapsulation of insulinoma cells: biotransport and biocompatibility considerations.

This study investigates whether nanoporous micromachined biocapsules, with uniform membrane pore sizes of 24.5-nm, can be used to encapsulate insulin-secreting cells in vitro. This approach to cell encapsulation is based on microfabrication technology whereby immunoisolation membranes are bulk and surface micromachined to present uniform and well-controlled pore sizes as small as 10 nm, tailored surface chemistries, and precise microarchitectures. This study evaluates the behavior of insulinoma cells with micromachined membranes, the effect of matrix configurations within the biocapsule on cell behavior, as well as insulin and glucose transport through the biocapsule membranes.

Evaluation of nanostructured composite collagen--chitosan matrices for tissue engineering.

The development of suitable three-dimensional matrices for the maintenance of cellular viability and differentiation is critical for applications in tissue engineering and cell biology. The structure and composition of the extracellular matrix (ECM) has been shown to modulate cell behavior with respect to shape, movement, proliferation, and differentiation. Although collagen and chitosan have separately been proposed as in vitro ECM materials, the influence of chitosan--collagen composite matrices on cell morphology, differentiation, and function is not well studied. To this end, gel matrices of different proportions of collagen and chitosan were examined ultrastructurally and characterized for their ability to regulate cellular activity. A three-chamber system with circulating hydraulic fluids was used to evaluate the gel stability under fluid force. Results indicated that overall matrix integrity increased with the proportion of chitosan. Scanning electron microscopy indicated that the addition of chitosan greatly influences ultrastructure and changes collagen fiber cross-linking, reinforcing the structure and increasing pore size. K562 cells cultured in three-dimensional gels were examined for cell proliferation and differentiation. Although cell proliferation was inhibited with an increasing proportion of chitosan, cell function based on cytokine-release was greatly augmented. Results suggest that a hybrid chitosan--collagen matrix may have potential biological and mechanical benefits for use as a cellular scaffold.

Micromachined interfaces: new approaches in cell immunoisolation and biomolecular separation.

As a novel therapeutic application of microfabrication technology, a micromachined membrane-based biocapsule is described for the transplantation of protein-secreting cells without the need for immunosuppression. This new approach to cell encapsulation is based on microfabrication technology whereby immunoisolation membranes are bulk and surface micromachined to present uniform and well-controlled pore sizes as small as 10 nm, tailored surface chemistries, and precise microarchitecture. Through its ability to achieve highly controlled microarchitectures on size scales relevant to living systems (from microm to nm), microfabrication technology offers unique opportunities to more precisely engineer biocapsules that allow free exchange of the nutrients, waste products, and secreted therapeutic proteins between the host (patient) and implanted cells, but exclude lymphocytes and antibodies that may attack foreign cells. Microfabricated inorganic encapsulation devices may provide biocompatibility, in vivo chemical and mechanical stability, tailored pore geometries, and superior immunoisolation for encapsulated cells over conventional encapsulation approaches. By using microfabrication techniques, structures can be fabricated with spatial features from the sub-micron range up to several millimeters. These multi-scale structures correspond well with hierarchical biological structures, from proteins and sub-cellular organelles to the tissue and organ levels.

Fabrication of microtextured membranes for cardiac myocyte attachment and orientation.

To understand the role of tissue adaptation to altered physiological states, a more physiologically and dimensionally relevant in vitro model of cardiac myocyte organization has been developed. A microtextured polymeric membrane with micron range dimensions promotes myocyte adhesion through substrate/cell interlocking and, thus, provides a more suitable stretchable matrix for studying overlying cell populations. These microtextured membranes are created using photolithography and microfabrication techniques. Biologically, mechanically, and optically compatible interfaces with specified microarchitecture and surface chemistry have been designed, microfabricated, and characterized for this purpose. Cardiac myocytes plated on these membranes display greater attachment and cell height compared to conventional culture substrates. Advantages of the microtextured membranes include the high degree of reproducibility and the ability to create features on the micron and submicron size scale. Because of the flexibility of substrate material and the ease of creating micron size structures, this technique can be applied to many other physiological and biological systems.

Nanoporous anti-fouling silicon membranes for biosensor applications.

The ability to create biocompatible well-controlled membranes has been an area of great interest over the last few years, particularly for biosensor applications. The present study describes the fabrication and characterization of novel nanoporous micromachined membranes that exhibit selective permeability and low biofouling. Results indicate that such membranes can be fabricated with uniform pore sizes capable of the simultaneous exclusion of albumin and diffusion of glucose. Compared to polymeric membranes of similar pore size, micromachined silicon membranes allowed more than twice the amount of glucose diffusion after 240 min and complete albumin exclusion. Moreover, membranes exhibit no morphological change or degradability in the presence of biological proteins and fluids at 37 degrees C. The results point to the potential of using such membranes for implantable biosensor applications. With monodisperse pores sizes as small as 10 nm, these membranes offer advantages in their reproducibility, stability, and ability to be integrated in silicon-based biosensing technology.

Microfabricated biocapsules provide short-term immunoisolation of insulinoma xenografts.

This study examines the viability and functionality of two insulinoma cell lines, RIN (1048) and betaTC6F7, encapsulated within microfabricated biocapsules. Surface and bulk micromachining are integrated in the biocapsule fabrication process, resulting in a diffusion membrane with uniform pore size distribution as well as mechanical and chemical stability, surrounded by an anisotropically-etched silicon wafer, which serves as the encapsulation cavity. Insulinoma cells (4500 cells/biocapsule) were enclosed within these microfabricated biocapsules and subjected to a static incubation study after either implantation in BALB-C mice or incubation in vitro. Examination of retrieved microfabricated biocapsules revealed an insulin stimulatory index of approximately 1.5 for encapsulated RIN cells and 3.6 for encapsulated betaTC6F7 cells for biocapsules with 18 nm pore sized microfabricated membranes, similar to indices of biocapsules incubated in vitro. There was an 80% decrease in cell stimulatory response between in vitro and in vivo 66 nm-biocapsules as compared to 20% for 18 nm-biocapsules, indicating that the immunoisolatory effectiveness depends greatly on achieving uniform pore sizes in the size range of 18 nm or smaller. The present study demonstrates the feasibility of using microfabricated biocapsules for the immunoisolation of insulinoma cells lines. The microfabricated biocapsule may serve as an alternative to conventional polymeric based biocapsules for possible use as in vivo insulin secreting bioreactor.

Microfabricated immunoisolating biocapsules.

A microfabricated silicon-based biocapsule for the immunoisolation of cell transplants is presented. The biocapsule-forming process employs bulk micromachining to define cell-containing chambers within single crystalline silicon wafers. These chambers interface with the surrounding biological environment through polycrystalline silicon filter membranes. The membranes are surface micromachined to present a high density of uniform pores, thus affording sufficient permeability to oxygen, glucose, and insulin. The pore dimensions, as small as 20 nm, are designed to impede the passage of immune molecules and graft-borne viruses. The underlying filter-membrane nanotechnology has been successfully applied in controlled cell culture systems (Ferrari et al., 1995), and is under study for viral elimination in plasma fractionation protocols. Here we report the encouraging results of in vitro experiments investigating the biocompatibility of the microfabricated biocapsule, and demonstrate that encapsulated rat neonatal pancreatic islets significantly outlive and outperform controls in terms of insulin-secretion capability over periods of several weeks. These results appear to warrant further investigations on the potential of cell xenografts encapsulated within microfabricated, immunoisolating environments for the treatment of insulin-dependent diabetes.

Lessons learned from the non-obese diabetic mouse II: Amelioration of pancreatic autoimmune isograft rejection during pregnancy.

To determine whether pregnancy provides an improved milieu for fetal/neonatal pancreas/islet transplantation, we studied neonatal pancreatic implants into non-obese diabetic (NOD) female mice during early gestation. We monitored maternal glycemic status, birthweight of the offspring, and graft histology to assess the efficacy of transplantation. One hundred and thirteen twelve-week-old NOD female mice were randomized into four groups as follows: (1) non-pregnant NOD mice received a sham operation; (2) non-pregnant NOD mice received neonatal pancreatic transplants; (3) pregnant NOD mice received a sham operation; and (4) pregnant NOD mice received neonatal pancreatic transplants. Pancreas segments from 3 neonatal NOD mice were placed via an incision 1 to 2 mm distal to the ear-skull junction of each of the recipients. Maternal blood glucose and glycated hemoglobin were determined between days 18 and 20 after the surgery. Pups were weighed within 5 to 6 hours after delivery. Pregnant NOD that received transplants (n = 29) had lower glucose and glycated hemoglobin (GHb) than sham operated pregnant controls (n = 26) (4.9 +/- 0.05 versus 9.0 +/- 5.0 mmol/L, p < 0.001 for glucose and 2.0 > or = 0.2 versus 3.0 > or = 1.2%, p < 0.008 for GHb) at 18 to 20 days of gestation. Controlling for litter size showed a decrease in birthweight for offspring of transplant recipients versus offspring of pregnant controls (1.59 +/- 0.08 versus 1.65 +/- 0.08 g, p < 0.002). Histological scoring of transplanted tissue at day 21 indicated that the lymphocytic infiltration in the pregnant group was significantly less than the control group (2.9 +/- 1.2 versus 4.9 +/- 0.2, p < 0.0001). We conclude that the pregnant NOD mouse provides a useful transplant model, that pregnancy provides an opportunity to increase beta-cell mass with transplanted tissue, and that pancreatic transplantation decrease birthweight and macrosomia in the offspring of NOD mice.