Rebecca L. Horan

Silk matrix for tissue engineered anterior cruciate ligaments

A silk-fiber matrix was studied as a suitable material for tissue engineering anterior cruciate ligaments (ACL). The matrix was successfully designed to match the complex and demanding mechanical requirements of a native human ACL, including adequate fatigue performance. This protein matrix supported the attachment, expansion and differentiation of adult human progenitor bone marrow stromal cells based on scanning electron microscopy, DNA quantitation and the expression of collagen types I and III and tenascin-C markers. The results support the conclusion that properly prepared silkworm fiber matrices, aside from providing unique benefits in terms of mechanical properties as well as biocompatibility and slow degradability, can provide suitable biomaterial matrices for the support of adult stem cell differentiation toward ligament lineages. These results point toward this matrix as a new option for ACL repair to overcome current limitations with synthetic and other degradable materials.

Cell differentiation by mechanical stress

Growth factors, hormones, and other regulatory molecules are traditionally required in tissue engineering studies to direct the differentiation of progenitor cells along specific lineages. We demonstrate that mechanical stimulation in vitro, without ligament‐selective exogenous growth and differentiation factors, induces the differentiation of mesenchymal progenitor cells from the bone marrow into a ligament cell lineage in preference to alternative paths (i.e., bone or cartilage cell lineages). A bioreactor was designed to permit the controlled application of ligament‐like multidimensional mechanical strains (translational and rotational strain) to the undifferentiated cells embedded in a collagen gel. The application of mechanical stress over a period of 21 days up‐regulated ligament fibroblast markers, including collagen types I and III and tenascin‐C, fostered statistically significant cell alignment and density and resulted in the formation of oriented collagen fibers, all features characteristic of ligament cells. At the same time, no up‐regulation of bone or cartilage‐specific cell markers was observed.

Advanced Bioreactor with Controlled Application of Multi-Dimensional Strain For Tissue Engineering

Advanced bioreactors are essential for meeting the complex requirements of in vitro engineering functional skeletal tissues. To address this need, we have developed a computer controlled bench-top bioreactor system with capability to apply complex concurrent mechanical strains to three-dimensional matrices independently housed in 24 reactor vessels, in conjunction with enhanced environmental and fluidic control. We demonstrate the potential of this new system to address needs in tissue engineering, specifically toward the development of a tissue engineered anterior cruciate ligament from human bone-marrow stromal cells (hBMSC), where complex mechanical and biochemical environment control is essential to tissue function. Well-controlled mechanical strains (resolution of < 0.1 micron for translational and < 0.1 degree for rotational strain) and dissolved oxygen tension (between 0%-95% +/- 1%) could be applied to the developing tissue, while maintaining temperature at 37 +/- 0.2 degrees C about developing tissue over prolonged periods of operation. A total of 48 reactor vessels containing cell culture medium and silk fiber matrices were run for up to 21 days under 90 degrees rotational and 2 mm translational deformations at 0.0167 Hz with only one succumbing to contamination due to a leak at an medium outlet port. Twenty-four silk fiber matrices seeded with human bone marrow stromal cells (hBMSCs) housed within reactor vessels were maintained at constant temperature (37 +/- 0.2 degrees C), pH (7.4 +/- 0.02), and pO2 (20 +/- 0.5%) over 14 days in culture. The system supported cell spreading and growth on the silk fiber matrices based on SEM characterization, as well as the differentiation of the cells into ligament-like cells and tissue (Altman et al., 2001).

Mechanical Stimulation Promotes Osteogenic Differentiation of Human Bone Marrow Stromal Cells on 3-D Partially Demineralized Bone Scaffolds In Vitro

Bone is a dynamic tissue that is able to sense and adapt to mechanical stimuli by modulating its mass, geometry, and structure. Bone marrow stromal cells (BMSCs) are known to play an integral part in bone formation by providing an osteoprogenitor cell source capable of differentiating into mature osteoblasts in response to mechanical stresses. Characteristics of the in vivo bone environment including the three dimensional (3-D) lacunocanalicular structure and extracellular matrix composition have previously been shown to play major roles in influencing mechanotransduction processes within bone cells. To more accurately model this phenomenon in vitro, we cultured human BMSCs on 3-D, partially demineralized bone scaffolds in the presence of four-point bending loads within a novel bioreactor. The effect of mechanical loading and dexamethasone concentration on BMSC osteogenic differentiation and mineralized matrix production was studied for 8 and 16 days of culture. Mechanical stimulation after 16 days with 10 nM dexamethasone promoted osteogenic differentiation of BMSCs by significantly elevating alkaline phosphatase activity as well as alkaline phosphatase and osteopontin transcript levels over static controls. Mineralized matrix production also increased under these culture conditions. Dexamethasone concentration had a dramatic effect on the ability of mechanical stimulation to modulate these phenotypic and genotypic responses. These results provide increased insight into the role of mechanical stimulation on osteogenic differentiation of human BMSCs in vitro and may lead to improved strategies in bone tissue engineering.

The Use of Long-term Bioresorbable Scaffolds for Anterior Cruciate Ligament Repair

The absence of adequate options to restore full knee joint function through anterior cruciate ligament reconstruction prompts the need to develop new ligament replacement strategies. Recent focus within the ligament engineering field has been on the establishment of appropriate anterior cruciate ligament graft design requirements and evaluation methods. A range of biomaterials and graft constructions has been explored in an attempt to identify the optimal ligament replacement. Thorough and standardized evaluation methods are required throughout all phases of development, from initial in vitro bench screening through a large animal in vivo model. The initial positive clinical, gross pathologic, histologic, and mechanical results from a 12-month in vivo goat study demonstrate the potential of bioengineered ligament devices.

An evaluation of SERI surgical scaffold for soft-tissue support and repair in an ovine model of two-stage breast reconstruction.

Summary: This study was designed to evaluate the SERI Surgical Scaffold, a silk-derived bioresorbable scaffold, in an ovine model of two-stage breast reconstruction. Sheep were implanted bilaterally with either SERI or sham sutures during the stage 1 procedure. The SERI group underwent an exchange procedure for a breast implant at 3 months; animals in the sham group were killed at 3 months. The sham samples were significantly weaker than the SERI plus tissue samples by 3 months. At all endpoints, SERI plus tissue samples were greater than or equal to 150 percent of native ovine fascial strength. Histologic evaluation of SERI samples showed evidence of bioresorption through 12 months. SERI provided adequate soft-tissue support with progressive bioresorption. By 12 months, newly formed tissue had assumed the majority of load-bearing responsibility.