George F. Muschler

Bone graft materials: An overview of the basic science

Autograft, allograft, and synthetic bone graft substitute materials play an important role in reconstructive orthopaedic surgery, and understanding the biologic effects of these materials is necessary for optimum use. Although vascularized and cancellous autograft show optimum skeletal incorporation, host morbidity limits autograft availability. Experimental studies have confirmed an immune response to allograft bone, but the clinical significance of this response in humans still is unclear. Small amounts of cancellous allograft in humans usually are remodeled completely; large allografts become incorporated by limited, surface intramembranous bone formation suggesting that these graft are primarily osteoconductive. Several synthetic skeletal substitute materials also are osteoconductive, and may show remodeling characteristics similar to allograft. Demineralized bone matrix and some isolated or synthetic proteins can induce endochondral bone formation, and therefore are osteoinductive. The extent and distribution of remodeling of bone graft materials are influenced by many factors, including the quality of the host site and the local mechanical environment (strain). Graft materials are likely to become more specialized for use in specific clinical applications, and composite preparations may soon provide bone graft materials with efficacy that equals or exceeds that of autogenous grafts.

Engineering principles of clinical cell-based tissue engineering.

Tissue engineering is a rapidly evolving discipline that seeks to repair, replace, or regenerate specific tissues or organs by translating fundamental knowledge in physics, chemistry, and biology into practical and effective materials, devices, systems, and clinical strategies. Stem cells and progenitors that are capable of forming new tissue with one or more connective tissue phenotypes are available from many adult tissues and are defined as connective tissue progenitors. There are four major cell-based tissue-engineering strategies: (1) targeting local connective tissue progenitors where new tissue is desired, (2) transplanting autogenous connective tissue progenitors, (3) transplanting culture-expanded or modified connective tissue progenitors, and (4) transplanting fully formed tissue generated in vitro or in vivo. Stem cell function is controlled by changes in stem cell activation and self-renewal or by changes in the proliferation, migration, differentiation, or survival of the progeny of stem cell activation, the downstream progenitor cells. Three-dimensional porous scaffolds promote new tissue formation by providing a surface and void volume that promotes the attachment, migration, proliferation, and desired differentiation of connective tissue progenitors throughout the region where new tissue is needed. Critical variables in scaffold design and function include the bulk material or materials from which it is made, the three-dimensional architecture, the surface chemistry, the mechanical properties, the initial environment in the area of the scaffold, and the late scaffold environment, which is often determined by degradation characteristics. Local presentation or delivery of bioactive molecules can change the function of connective tissue progenitors (activation, proliferation, migration, differentiation, or survival) in a manner that results in new or enhanced local tissue formation. All cells require access to substrate molecules (oxygen, glucose, and amino acids). A balance between consumption and local delivery of these substrates is needed if cells are to survive. Transplanted cells are particularly vulnerable. Theoretical calculations can be used to explore the relationships among cell density, diffusion distance, and cell viability within a graft and to design improved strategies for transplantation of connective tissue progenitors. Rational strategies for tissue engineering seek to optimize new tissue formation through the logical selection of conditions that modulate the performance of connective tissue progenitors in a graft site to produce a desired tissue. This increasingly involves strategies that combine cells, matrices, inductive stimuli, and techniques that enhance the survival and performance of local or transplanted connective tissue progenitors.

Age- and gender-related changes in the cellularity of human bone marrow and the prevalence of osteoblastic progenitors.

Bone marrow harvested by aspiration contains connective tissue progenitor cells which can be induced to express a bone phenotype in vitro. The number of osteoblastic progenitors can be estimated by counting the colony‐forming units which express alkaline phosphatase (CFU‐APs). This study was undertaken to test the hypothesis that human aging is associated with a significant change in the number or prevalence of osteoblastic progenitors in the bone marrow. Four 2‐ml bone marrow aspirates were harvested bilaterally from the anterior iliac crest of 57 patients, 31 men (age 15–83) and 26 women (age 13–79). A mean of 64 million nucleated cells was harvested per aspirate. The mean prevalence of CFU‐APs was found to be 55 per million nucleated cells. These data revealed a significant age‐related decline in the number of nucleated cells harvested per aspirate for both men and women (P = 0.002). The number of CFU‐APs harvested per aspirate also decreased significantly with age for women (P = 0.02), but not for men (P = 0.3). These findings are relevant to the harvest of bone marrow derived connective tissue progenitors for bone grafting and other tissue engineering applications, and may also be relevant to the pathophysiology of age‐related bone loss and post‐menopausal osteoporosis.

Aspiration to Obtain Osteoblast Progenitor Cells from Human Bone Marrow: The Influence of Aspiration Volume*

Bone marrow contains osteoblast progenitor cells that can be obtained with aspiration and appear to arise from a population of pluripotential connective-tissue stem cells. When cultured in vitro under conditions that promote an osteoblastic phenotype, osteoblast progenitor cells proliferate to form colonies of cells that express alkaline phosphatase and, subsequently, a mature osteoblastic phenotype. We evaluated the number of nucleated cells in bone-marrow samples obtained with aspiration from the anterior iliac crest of thirty-two patients without systemic disease. There were nineteen male patients and thirteen female patients; the mean age was forty-one years (range, fourteen to seventy-seven years). The prevalence and concentration of the osteoblast progenitor cells also were determined, by placing the bone-marrow-derived cells into tissue-culture medium and counting the number of alkaline phosphatase-positive colony-forming units. In order to assess the effect of aspiration volume, two sequential experiments were performed. In the first experiment, aspiration volumes of one and two milliliters were compared. In the second experiment, aspiration volumes of two and four milliliters were compared. The mean prevalence of alkaline phosphatase-positive colony-forming units in the bone-marrow samples was thirty-six per one million nucleated cells (95 per cent confidence interval, 28 to 47); a mean of 2400 alkaline phosphatase-positive colony-forming units was obtained from a two-milliliter aspirate. There was a significant difference among the patients with respect to the number of alkaline phosphatase-positive colony-forming units in these bone-marrow samples (p < 0.001). Seventy per cent of this variation in the prevalence was due to variation among patients, and 20 per cent was due to variation among aspirates. The number of alkaline phosphatase-positive colony-forming units in the aspirate increased as the aspiration volume increased. However, contamination by peripheral blood also increased as the aspiration volume increased. An increase in the aspiration volume from one to four milliliters caused a decrease of approximately 50 per cent in the final concentration of alkaline phosphatase-positive colony-forming units in an average sample. CLINICAL RELEVANCE: On the basis of these data, we recommend that, when bone marrow is obtained with aspiration for use as a bone graft, the volume of aspiration from any one site should not be greater than two milliliters. A larger volume decreases the concentration of osteoblast progenitor cells because of dilution of the bone-marrow sample with peripheral blood. We estimate that four one-milliliter aspirates will provide almost twice the number of alkaline phosphatase-positive colony-forming units as will one four-milliliter aspirate. In addition, these data confirm that humans differ significantly from one another with respect to the cellularity of bone marrow and the prevalence of osteoblast progenitor cells. Additional studies are necessary to determine if the number or prevalence of alkaline phosphatase-positive colony-forming units in bone marrow is a determining factor in the efficacy of an autogenous bone or bone-marrow graft and to ascertain how the number and function of alkaline phosphatase-positive colony-forming units may change as a function of factors such as age, menopausal status, and selected diseases.

BONE CELLS AND MATRICES IN ORTHOPEDIC TISSUE ENGINEERING

The ability to harvest and manipulate osteogenic cells gives clinicians the opportunity to harness capacity of these cells for targeted regeneration and repair of skeletal tissues. Further opportunities to optimize use of cells exist in the ability to design specialized matrices that act as conductive scaffolds. Realization of the full potential of engineered matrix materials and cell-matrix composites can provide new solutions to many clinical problems in skeletal reconstruction.

Spine Fusion Using Cell Matrix Composites Enriched in Bone Marrow-Derived Cells

Bone marrow-derived cells including osteoblastic progenitors can be concentrated rapidly from bone marrow aspirates using the surface of selected implantable matrices for selective cell attachment. Concentration of cells in this way to produce an enriched cellular composite graft improves graft efficacy. The current study was designed to test the hypothesis that the biologic milieu of a bone marrow clot will significantly improve the efficacy of such a graft. An established posterior spinal fusion model and cancellous bone matrix was used to compare an enriched cellular composite bone graft alone, bone matrix plus bone marrow clot, and an enriched bone matrix composite graft plus bone marrow clot. Union score, quantitative computed tomography, and mechanical testing were used to define outcome. The union score for the enriched bone matrix plus bone marrow clot composite was superior to the enriched bone matrix alone and the bone matrix plus bone marrow clot. The enriched bone matrix plus bone marrow clot composite also was superior to the enriched bone matrix alone in fusion volume and in fusion area. These data confirm that the addition of a bone marrow clot to an enriched cell-matrix composite graft results in significant improvement in graft performance. Enriched composite grafts prepared using this strategy provide a rapid, simple, safe, and inexpensive method for intraoperative concentration and delivery of bone marrow-derived cells and connective tissue progenitors that may improve the outcome of bone grafting.

Connective tissue progenitors: practical concepts for clinical applications.

Tissue engineering can be defined as any effort to create or induce the formation of a specific tissue in a specific location through the selection and manipulation of cells, matrices, and biologic stimuli. The biologic concepts and the biochemical and biophysical principles on which these efforts are based have become an exciting and rapidly evolving field of biomedical research. More importantly, tissue engineering is becoming a clinical reality in the practice of orthopaedic surgery, providing patients and physicians with an expanding set of practical tools for effective therapy. New and improved matrices and bioactive factors inevitably will play important roles in the evolution of orthopaedic tissue engineering. However, tissue engineering never can stray far from fundamental biologic principles, and one of these is that cells do all the work. No new tissue forms except through the activity of living cells. No bone graft, no matrix, no growth factor, no cytokine can contribute to the generation or integration of new tissue, except through the influence it has on the behavior of cells. The efficacy of all current clinical tools depends entirely on the cells in the grafted site, particularly the small subset of stem cells and progenitor cells that are capable of generating new tissue. The current authors review a series of key biologic concepts related to the rational design and selection of composites of cells and matrices in contemporary bone grafting and tissue engineering efforts. The functional paradigms of stem cell biology are reviewed, including self renewal, asymmetric and symmetric mitosis, and lineage restriction. Several potential sources for autogenous stem cells for connective tissues are discussed. Finally, a simple mathematical model is introduced as a tool for understanding the functional demands placed on stem cells and progenitors in a graft site and to provide a conceptual framework for the rational design of cell matrix composite grafts.

Evaluation of collagen ceramic composite graft materials in a spinal fusion model

Autogenous bone graft is highly effective in inducing a bone healing response in most clinical settings. However, significant morbidity can occur related to the harvest of an autograft. This makes the development of synthetic or purified nontissue bone grafting materials highly desirable. Both purified bovine Type I collagen and calcium phosphate ceramics have been proposed as promising osteoconductive bone graft substitute materials. One collagen ceramic composite, Collagraft, is approved for use in acute long bone fractures. This study evaluated composites of purified bovine Type I fibrillar collagen and a granular biphasic hydroxyapatite/tricalcium phosphate ceramic in the posterior segmental canine spinal fusion model. Materials were compared based on union score and mechanical testing in 3 separate fusion sites (L1-2, L3-4, L5-6). All composites were found to be inferior in union score to an equal volume of autogenous cancellous bone. In addition, the combination of the collagen ceramic composite with autogenous cancellous bone graft reduced the effectiveness of the autogenous bone graft significantly. These data should be a caution to the clinician who may consider use of collagen ceramic composites similar to Collagraft for spinal fusion applications.

Selective retention of bone marrow-derived cells to enhance spinal fusion.

Connective tissue progenitors can be concentrated rapidly from fresh bone marrow aspirates using some porous matrices as a surface for cell attachment and selective retention, and for creating a cellular graft that is enriched with respect to the number of progenitor cells. We evaluated the potential value of this method using demineralized cortical bone powder as the matrix. Matrix alone, matrix plus marrow, and matrix enriched with marrow cells were compared in an established canine spinal fusion model. Fusions were compared based on union score, fusion mass, fusion volume, and by mechanical testing. Enriched matrix grafts delivered a mean of 2.3 times more cells and approximately 5.6 times more progenitors than matrix mixed with bone marrow. The union score with enriched matrix was superior to matrix alone and matrix plus marrow. Fusion volume and fusion area also were greater with the enriched matrix. These data suggest that the strategy of selective retention provides a rapid, simple, and effective method for concentration and delivery of marrow-derived cells and connective tissue progenitors that may improve the outcome of bone grafting procedures in various clinical settings.

A three dimensional scaffold with precise micro-architecture and surface micro-textures

A three-dimensional (3D) structure comprising precisely defined micro-architecture and surface micro-textures, designed to present specific physical cues to cells and tissues, may provide an efficient scaffold in a variety of tissue engineering and regenerative medicine applications. We report a fabrication technique based on microfabrication and soft lithography that permits for the development of 3D scaffolds with both precisely engineered architecture and tailored surface topography. The scaffold fabrication technique consists of three key steps starting with microfabrication of a mold using an epoxy-based photoresist (SU-8), followed by dual-sided molding of a single layer of polydimethylsiloxane (PDMS) using a mechanical jig for precise motion control; and finally, alignment, stacking, and adhesion of multiple PDMS layers to achieve a 3D structure. This technique was used to produce 3D Texture and 3D Smooth PDMS scaffolds, where the surface topography comprised 10 microm diameter/height posts and smooth surfaces, respectively. The potential utility of the 3D microfabricated scaffolds, and the role of surface topography, were subsequently investigated in vitro with a combined heterogeneous population of adult human stem cells and their resultant progenitor cells, collectively termed connective tissue progenitors (CTPs), under conditions promoting the osteoblastic phenotype. Examination of bone-marrow derived CTPs cultured on the 3D Texture scaffold for 9 days revealed cell growth in three dimensions and increased cell numbers compared to those on the 3D Smooth scaffold. Furthermore, expression of alkaline phosphatase mRNA was higher on the 3D Texture scaffold, while osteocalcin mRNA expression was comparable for both types of scaffolds.