Gregory H. Altman

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.

Human bone marrow stromal cell responses on electrospun silk fibroin mats.

Fibers with nanoscale diameters provide benefits due to high surface area for biomaterial scaffolds. In this study electrospun silk fibroin-based fibers with average diameter 700+/-50 nm were prepared from aqueous regenerated silkworm silk solutions. Adhesion, spreading and proliferation of human bone marrow stromal cells (BMSCs) on these silk matrices was studied. Scanning electron microscopy (SEM) and MTT analyses demonstrated that the electrospun silk matrices supported BMSC attachment and proliferation over 14 days in culture similar to native silk fibroin (approximately 15 microm fiber diameter) matrices. The ability of electrospun silk matrices to support BMSC attachment, spreading and growth in vitro, combined with a biocompatibility and biodegradable properties of the silk protein matrix, suggest potential use of these biomaterial matrices as scaffolds for tissue engineering.

Macrophage responses to silk.

Silk fibers have potential biomedical applications beyond their traditional use as sutures. The physical properties of silk fibers and films make it a promising candidate for tissue engineering scaffold applications, particularly where high mechanical loads or tensile forces are applied or in cases where low rates of degradation are desirable. A critical issue for biomaterial scaffolds is biocompatibility. The direct inflammatory potential of intact silk fibers as well as extracts was studied in an in vitro system. The results indicate that silk fibers are largely immunologically inert in short- and long-term culture with RAW 264.7 murine macrophage cells while insoluble fibroin particles induced significant TNF release. Soluble sericin proteins extracted from native silk fibers did not induce significant macrophage activation. While sericin did not activate macrophages by itself, it demonstrated a synergistic effect with bacterial lipopolysaccharide. The low level of inflammatory potential of silk fibers makes them promising candidates in future biomedical applications.

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).

Matrix metalloproteinases and their clinical applications in orthopaedics.

Imbalance in the expression of matrix metalloproteinases and their inhibitors contribute considerably to abnormal connective tissue degradation prevalent in various orthopaedic joint diseases such as rheumatoid arthritis and osteoarthritis. Matrix metalloproteinase expression has been detected in ligament, tendon, and cartilage tissues in the joint. They are known to contribute to the development, remodeling, and maintenance of healthy tissue through their ability to cleave a wide range of extracellular matrix substrates. Their role has been extended to cell growth, migration, differentiation, and apoptosis. In orthopaedics, their clinical applications constantly are being explored. The multiple steps in matrix metalloproteinase regulation offer potential targets for inhibition, useful in drug therapy. The correlation between matrix metalloproteinases and progression in joint erosion presents potential prognostic and diagnostic tools in rheumatoid arthritis. Matrix metalloproteinases also can be incorporated into scaffold design to control the degradation rate of engineered tissue constructs. This current review aims to summarize and emphasize the importance of matrix metalloproteinases and their natural inhibitors in the maturation of musculoskeletal tissue through matrix remodeling and, therefore, in the generation of a new clinical potential in orthopaedics.

Future direction of the treatment of ACL ruptures

The future of treatment of the ACL rupture is changing as our understanding of the biology surrounding the ACL continues to increase. It is our expectation that clinically applicable treatments, including the repair of the ACL and the development of a biologically engineered ACL, will occur in the next decade.

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.

Characterization of transcript levels for matrix molecules and proteases in ruptured human anterior cruciate ligaments.

An improved understanding of cellular responses during normal anterior cruciate ligament (ACL) function or repair is essential for clinical assessments, understanding ligament biology, and the implementation of tissue engineering strategies. The present study utilized quantitative real-time RT-PCR combined with univariate and multivariate statistical analyses to establish a quantitative database of marker transcript expression that can provide a “blueprint” of ACL wound healing. Selected markers (collagen types I and III, biglycan, decorin, MMP-1, MMP-2, MMP-9, and TIMP-1) were assessed from 33 torn ACLs harvested during reconstructive surgery. Trends were observed between postinjury period and marker expressions. Significant correlations between marker expression existed and were most prominent between collagen types I and III. Canonical correlation analysis established a relationship between patient demographics and a combination of all marker expressions. The currently observed trends and correlations may assist in identifying appropriate tissue samples and provide a baseline information of marker expression level that can support in vitro optimization of environmental cues for ligament tissue engineering application.

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.