Contraction is the principal function of muscle cells and is necessary for survival. Contraction of cardiac muscle cells enables the pumping action of the heart; skeletal muscle cell contraction facilitates locomotion; and the smooth muscle cell contraction controls the constriction of vessels. Under certain circumstances, however, connective tissue cells can also con-tract by expressing genes for the contractile cytoskeletal proteins normally found in muscle cells. Recent work in the Orthopaedic Research Laboratory (ORL) at the Brigham and Women’s Hospital has demonstrated that the connective tissue cells comprising many musculoskeletal tissues can synthesize muscle actin, and can display contractile behavior under cer-tain circumstances.

This data has led us to consider the following important questions: (1) What roles could contraction play in the func-tion of a fibroblast, chondrocyte, or osteoblast in the processes of tissue formation and remodeling or in wound healing? (2) Can such contraction contribute to pathological conditions in the musculoskeletal tissues that necessitate orthopaedic care? (3) Could therapeutic regulation of the contractile process in these cells enable the prevention of certain disorders? (4) How will contractile behavior impact tissue-engineering approaches? Work in progress in the ORL is attempting to answer these and other relevant questions.

Investigations of the expression of a contractile actin iso-form, á -smooth muscle actin (SMA), by connective tissue cells and evaluation of the contractility of the cells in vitro have been proceeding in the ORL for over five years. The initial study in this line of investigation dealt with the healing of the rabbit medial collateral ligament (MCL). Motivated by the work of Professor I.V. Yannas at MIT on skin wound contraction, we investigated the presence of SMA-containing fibroblasts in the MCL during healing. Our initial study employed an immunohistochemical method to identify cells containing this actin isoform. Once we began to identify SMA-positive cells in the heal-ing ligament, we established a collaboration with Giulio Gabbiani, M.D., in Geneva, Switzerland. Dr. Gabbiani is credited with discovering the myofibroblast, in the early 1970's 7 . As part of this collaboration, immunohistochemistry, Western blot analysis, and transmission electron microscopy (TEM) were performed both in the ORL and in Dr. Gabbiani's laboratory (6) .

Dr. Spector is Director of Orthopaedic Research at Brigham
and Women’s Hospital,and Professor of Orthopaedic Surgery
(Biomaterials) at Harvard-M.I.T. Division of
Health Sciences and Technology

Please address correspondence to:

Myron Spector, PhD
Department of Orthopaedic Surgery
Brigham and Women’sHospital
75 Francis St
Boston, MA 02115
mspector@rics.bwh.harvard.edu

Recently, we have reported the presence of SMA in musculoskeletal connective tissue cells using immunohistochemistry. Cells studied include human anterior cruciate ligament fibrob-lasts (16) ; bovine (15) and human (1) meniscus cells; canine (22) and human (8) intervertebral disc cells; canine (10, 25) and human (9, 19) articular chondrocytes; and human osteoblasts 14 . In addition we have reported the presence of SMA-containing cells in the healing rat sciatic nerve (3, 4) and the rabbit Achilles tendon (11) , and in chondrocytic cells in healing defects in canine articular cartilage (25) . Western blot analysis has confirmed the presence of SMA in chondrocytes isolated from canine articular cartilage (19) . In related studies we have demonstrated the capability of SMA-containing meniscus cells (15) , intervertebral disc cells (22) , tendon cells (23), articular chondrocytes (10) and osteoblasts (14) to contract a collagenglycosaminoglycan (GAG) analog of extracellular matrix in vitro. These studies have demonstrated the potential of connective tissue cells comprising musculoskeletal tissue for contraction. This had led us to direct our attention to the role of contractile behavior in the formation, remodeling and healing of these tissues and on tissue engineering approaches being implemented for their regeneration. Specifically, the contractile behavior of connective tissue cells (i.e., nonmuscle) cells of the musculoskeletal tissues may contribute to the following:

a. Movement of extracellular matrix molecules in the process of imparting the specific architecture to the tissue.

b. Production of in situ strain (as in ligament).

c. Contraction of reparative tissue to facilitate wound closure.

d. Contracture of hyperplastic tissues occurring during healing or as a result of disease.

e. Retraction of the ends of ruptured tissue in which reparative tissue has not formed.

f. Contracture of compliant scaffolds employed as matrices for tissue engineering.

While some of the contractile functions may have a posi-tive influence on remodeling and healing (e.g., a, b, and c above), other behavior may not (e.g., d, e, and f). The control of the expression of contractile cytoskeletal elements in mus-culoskeletal connective tissue cells (e.g., fibroblasts, chondro-cytes, and osteoblasts) may be found to be of therapeutic ben-efit in modulating healing and disease processes.

INITIAL WORK REVEALING THE CONTRACTILE POTENTIAL OF FIBROBLASTS

Since the early 1900s investigators have considered the contractile behavior of nonmuscle cells in the processes underlying closure of wounds (viz., skin wounds) (21) . Propositions that this closure was not reliant on cells, but rather due to changes in the collagen of the extracellular matrix, were dispelled by studies showing that wound closure occurred in animals in which collagen synthesis and organization were impaired. It is now widely recognized that certain connective tissues undergo cell-mediated contraction. The contraction of healing skin wounds and pathological contractures such as Dupuytren's are two of the most striking examples. Cell-mediated tissue contraction can have both beneficial effects, such as in closure of a wound, and adverse effects, such as those demonstrated by the loss of function due to pathological contractures.

Two mechanisms by which cells such as fibroblasts exert a force on the matrix to which they are adherent have been proposed: 1) the cells pull on the matrix as they migrate, in a process that has been referred to as "tractional structuring" or "tractional remodeling" (5) ; or 2) the cells contract. Evidence that contraction of nonmuscle cells (viz., fibroblasts) may, at least in part, be the cause of tissue contracture include: a) the TEM demonstration of the presence of intracellular microfila-ments (7-9nm in diameter) - like those that can be seen in smooth muscle cells - arrayed along the length of the cells, suitably arranged for cell contraction; b) the biomechanical demonstration that agents known to stimulate smooth muscle cells cause the contraction in vitro of granulation tissue explants predominantly comprising fibroblasts with ultrastructural features of smooth muscle cells; and c) the immunohistochemical finding that fibroblasts in several contracting tissues expressed the gene for muscle actin(viz., SMA) previously observed only in smooth muscle cells and pericytes. These cells also demonstrated typical fibroblastic features along with those of smooth muscle cells, and this has led to the terms "myofibroblast" and "contractile fibroblasts."

There are six actin isoforms, coded by different genes. Most cells have the â and ã isoforms associated with the main-tenance of cell shape and cell migration. The four muscle actins include: á -smooth muscle (vascular), á -skeletal muscle, á -cardiac muscle, and ã -smooth muscle (enteric). While it is, of course, desirable to demonstrate several of the contractile- associated characteristics of cells prior to identifying them as myofibroblasts, there is some agreement among many that the presence of SMA alone in fibroblasts is enough to warrant their being classified as a myofibroblast (12) . Moreover, it has been noted that the majority of myofibroblasts express, albeit transiently, SMA, but not smooth muscle myosin and desmin. More recent work (2) has concluded that fibroblast contraction of collagen lattices, as investigated in vitro, is "dependent on á -sm actin expression and that á -sm actin is a functional marker for a fibroblast subtype that rapidly remodels the extracellular matrix."

While the role of non-muscle contractile cells in skin wound healing and certain pathological contractures has been studied for over 25 years, the contribution of these cells to the embryological and postnatal development of tissues and organs and to maintenance of the extracellular matrix archi-tecture in the adult are only now being investigated. The ORL has been engaged in studies to determine the presence of con-tractile cells in the musculoskeletal tissues and to assess their role in remodeling, the response to injury and healing, and in tissue engineering approaches to the orthopaedic treatment of disorders.

SMOOTH MUSCLE ACTIN - CONTAINING CELLS IN MUS - CULOSKELETAL CONNECTIVE TISSUES AND NERVE

Cells with myofibroblastic ultrastructural features and the SMA phenotype have been identified in certain developing, normal adult, and pathological connective tissues (24) for many years. However, there have been few reports of these cells in musculoskeletal tissues. Recent work from the ORL has now demonstrated the presence of this contractile phenotype in many of the musculoskeletal tissues.

Ligaments, the Meniscus, and Intervertebral Disc

The initial finding of SMA-containing fibroblasts in the rabbit MCL (6) led to the proposition that the contractile behavior of the cells may be responsible for reparative tissue contracture and re-establishment of the in situ strain normally occurring in the ligament, and found to be recovered to some extent after healing. This work was followed more recently by an immunohistochemical investigation of the presence of SMA-expressing cells in the intact human ACL (in nonvascular cells). We recently reported that the anteromedial bundle of human ACLs obtained during total knee arthroplasty contained 10-20% of cells with this particular actin isoform (16) . It was proposed that the contractile cells may contribute to the crimp-like architecture of the tissue. Also of interest was the subsequent finding of numerous SMA-containing cells in the epiligamentous tissue and synovial layer on the surface of rup-tured human ACLs (13) . One supposition was that the contraction of these cells might be responsible for the retraction of the remnants of the ruptured human ACL observed clinically.

We have also recently found SMA-positive cells in the calf (15) and human (1) meniscus and canine (22) and human (8) intervertebral disk. Moreover, we reported that when these cells were isolated from the tissues and seeded into collagen-GAG scaffolds they were able to contract the matrix (15, 22) . The contractile actin isoform was isolated from approximately 10-30 % of the cells in these tissues. Of note, many of the SMA-positive cells had the morphology of chondrocytes (also referred to as "fibrochon-drocytes") and were found in lacunae.

SMA-Containing Articular Chondrocytes

We have recently completed an immunohistochemical study (9) showing that a majority of chondrocytes in certain regions of human articular cartilage contained SMA. The percentage of SMA-positive chondrocytes in the superficial region of human samples was 73±3% (mean±SEM) with a range of 37%-96%. In contrast, only 11±2% of the chondrocytes in the deep region of articular cartilage stained positive for this con-tractile isoform (range 0-29%). There was a statistically significant difference in the percentage of SMA-positive cells in the superficial and deep regions (Student’s t-test: p<0.0001). The Spearman rank correlation test did not show a significant correlation of the percentage of SMA-positive cells in the superfi-cial region or the deep region with the Mankin grade of osteoarthritis. Moreover, there was no correlation of the percentage of SMA-containing cells in either region with patient age.

The objective of another recent study (25) was to evaluate the types of tissue resulting from spontaneous healing of surgically created defects in adult canine articular cartilage up to 29 weeks postoperatively, with specific attention directed toward the presence and distribution of cells containing SMA. Two 4-mm diameter defects were made in the trochlear groove to the depth of the tidemark in twenty adult mongrel dogs. Approximately 50% of the cells in the superficial layer of the uninvolved articular cartilage, beyond 1mm from the border of the defect, contained SMA while approximately one quarter of the cells in the deep layer displayed this contractile actin isoform. The difference in the percentage of SMA-positive chondrocytes in these two zones was statistically significant (p<0.003). There was no significant effect of postoperative time on the percentage of SMA-containing chondrocytes in any region of articular carti-lage surrounding the defect. Also of interest was that a greater percentage of chondrocytes in the deep layer adjacent to the defect (injury zone; 35 ± 5%) than those in the uninvolved deep zone (23 ± 5%) contained SMA (p=0.08).

SMA was also found in a large percentage of the cells comprising hyaline cartilage, fibrocartilage, and fibrous tissue in the defects after 6 to 16 weeks. Of interest was the fact that a greater percentage of cells in the cartilaginous tissues contained the contractile actin isoform, compared to the fibrob-lasts comprising the fibrous tissue. There was an increase in the percentage of SMA-positive cells in the fibrocartilage with time, with almost 75% of the cells containing SMA after 16 weeks. To some extent the finding of SMA-containing cells in reparative tissue in a cartilage defect parallels the initial finding of SMA-containing fibroblasts at certain stages of healing of skin wounds. In the case of skin wounds it has been proposed that contraction of myofibroblasts facilitates wound closure. Future work needs to be directed at determining the role of SMA in the reparative tissue in cartilage lesions.

Another recent study investigated the contractile behavior of cultured canine chondrocytes seeded in collagen-GAG scaffolds. Chondrocytes isolated from the knee joints of adult canines by collagenase digestion and expanded in monolayer culture were seeded into porous collagen-GAG scaffolds. Scaffolds were of two different compositions, with the predominant collagen being either type I or type II collagen, and of varying pore diameters. Over the four-week culture period, the seeded cells contracted all of the type I and type II collagen-based matrices, despite a wide range in stiffness (145 ± 23 Pa, for the type I scaffold, to 732 ± 35 Pa, for the type II material). Pore diameter (25-85 µm, type I; and 53-257 µm, type II) did not affect cell-mediated contraction. Light microscopy revealed that cells were distributed throughout the matrices. However, pores at the outer edge of the matrices were noticeably more compressed than inner pores. Immunohistochemical staining for the SMA isoform revealed that a majority of the chondrocytes seeded in the matrices contained this contractile actin.

It has been well documented that during growth in two-dimensional culture, chondrocytes adopt many of the phenotypic traits of fibroblasts, becoming elongated and synthesizing type I rather than type II collagen. The re-expression of the chondrocyte phenotype has been shown to occur when the cells are subsequently introduced into certain culture conditions (e.g., agarose gels). Using matrices similar to those used here, one of our prior investigations (17) found that while the majority of the cultured chondrocytes seeded in the type I collagen matrices had a fibroblastic morphology, the majority of the cells in the type II collagen matrices had a chondrocyte morphology and displayed an increase in GAG production, indicating, perhaps, that the cells are able to redifferentiate in the type II collagen matrix. Despite the predominance of cells with the chondrocyte morphology in the type II scaffolds, this study found that both the type I and type II matrices underwent cell-mediated contraction.

The expression of SMA is not merely the result of the dedifferentiation of chondrocytes to fibroblasts. Of importance is that immunohistochemical staining for SMA demonstrated that the articular cartilage chondrocytes in situ contain this contractile actin. When these cells were isolated and expanded in vitro, they continued to express this isoform and were capable of contracting an extracellular matrix.

Osteoblasts

Immunohistochemistry recently revealed the presence of SMA in some cells in human and canine trabecular bone spec-imens (14) . The majority of the cells of an osteoblastic cell line, MC3T3-E1, also expressed this actin isoform in two-dimen-sional culture and in a collagen-GAG matrix. These SMA-containing cells were found to cause contraction of the analog of extracellular matrix. The presence of SMA in the MC3T3-E1 cells was confirmed by Western blot analysis. Osteoblast con-tractile behavior might help to explain the mechanism by which they impart architecture to bone matrix, including that at implant interfaces. Moreover, the contraction of osteoblasts in situ may relate to their retraction on the bone surface to reveal bone matrix for osteoclast resorption and thus be a critical aspect of bone remodeling. In matrices implemented for bone tissue engineering, the contraction of osteoblasts may distort the scaffold and cause collapse of the pores thereby jeopardizing the performance of the implant. An understanding of this process could also guide the development of matrices for bone tissue engineering.

Contractile Fibroblasts in Peripheral Nerve

The fibrocollagenous connective tissues, comprising the endoneurium, perineurium, and epineurium of peripheral nerves provide structural support and isolate the nerve fibers from surrounding tissues. Thirty years ago, Ross and Reith (20) proposed that the perineurium also had contractile properties that might allow it to function to actively shorten the nerve to adjust for body movement. Their hypothesis was supported by their TEM findings of perineurial fibroblastic cells with the ultrastructural features of smooth muscle cells in the mouse sciatic nerve.

The initial finding of fibroblasts with ultrastructural features of smooth muscle cells in the perineurium of normal nerves was followed in later years by TEM studies of healing peripheral nerve that identified myofibroblasts in the reparative tissue in synthetic tubes bridging gaps experimentally produced in the rat sciatic nerve after 7-12 days. More recently, the contraction of the cellular bridge that formed between the stumps of severed peripheral nerves in mice after 7-8 days has been attributed to the fibroblasts within the reparative connective tissue. It was suggested that, in addition to mechanically pulling the nerve stumps together, the forces exerted by these cells might align the collagen fibrils in their newly deposited extracellular matrix and thereby provide guidance for the elongating axons toward the distal stump. These findings raise the question of the role of contractile cells in the connective tissue on healing of peripheral nerve.

In our own studies of peripheral nerve regeneration through tubular devices in a rat model, in addition to finding SMA-containing fibroblasts within the reparative tissue we found a thicker layer of SMA-containing fibroblasts bordering silicone tubes than collagen tubes (3, 4) . We proposed that the contractile forces exerted by the myofibroblasts within the reparative tissue may act on axons to facilitate their growth (3) . However, we have also noted that myofibroblasts bordering the inner wall of the tubular device when contracting circumferentially could be acting to constrict the formation of nervous tissue within the lesion (4) .

SUMMARY

Current work in the ORL has shown that a variety of musculoskeletal connective tissue cells can express the gene for SMA, and can contract an analog of extracellular matrix in vitro. The presence of SMA alone in a fibroblast has been considered sufficient, although not necessary in the presence of certain ultrastructural features demonstrated by TEM, for the identification of the cell as a myofibroblast. It had previously been proposed that myofibroblasts derive from three different cell types: smooth muscle cells, pericytes, and fibroblasts. The presence SMA-containing cells in articular cartilage with typical histological features of chondrocytes has raised the ques-tion of whether they should be referred to as "myochondro-cytes." The finding in a prior study that some chondroblasts contained SMA prompted those authors to suggest that such cells might be referred to "myochondroblasts" (18) . However, it is becoming increasing clear that a wide variety of cell types can express the gene for SMA (in some cases transiently) under certain in vivo and in vitro circumstances. This raises the question of whether it is necessary or meaningful to refer to these cells with the prefix "myo-."

The recent studies that have shown the presence of SMA-containing cells in musculoskeletal tissues in situ have laid the foundation for future investigations to determine the specific roles of these cells with contractile potential. Moreover, regu-lation of the contractile behavior of these cells may be found to be important in the treatment of certain disorders and in mus-culoskeletal tissue engineering approaches.