Macrophages accumulate in the early phase of tendon–bone healing

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Abstract

A scar tissue interface forms rather than a normal ligament insertion site following attachment of a tendon graft to bone. The specific cell types that initiate the process of tendon-to-bone healing are unknown. We hypothesized that inflammatory cell accumulation following tendon-to-bone repair results in this scar interface. We used a rodent model to examine the temporal and spatial pattern of accumulation of hematopoietic lineage cells in the early phase of tendon-to-bone healing. Thirty-six Lewis rats underwent anterior cruciate ligament (ACL) reconstruction in the left knee using a flexor digitorum longus tendon graft. Six animals were sacrificed at 4, 7, 11, 14, 21, and 28 days after surgery. Serial sections were analyzed for proliferating cells (PCNA), recruited macrophages (ED1), resident macrophages (ED2), neutrophils, T-lymphocytes (CD3), mast cells, immature progenitor cells/pericytes (expressing the NG2 cell-surface chondroitin sulfate proteoglycan), and newly-formed blood vessels (Factor VIII). Neutrophils, ED1+ and ED2+ macrophages accumulated sequentially in the healing tendon graft, with progressive cell ingrowth from the interface towards the inner tendon. Neutrophils and ED1+ cells were seen in the tendon–bone interface at 4 days after surgery, while ED2+ macrophages were not identified until 11 days. These cells progressively repopulated the tendon graft. NG2-positive progenitor cells were found along the edge of the bone tunnel in the interface, but these cells did not invade the tendon. Occasional T-lymphocytes and mast cells were seen in the tendon–bone interface. There was no proliferation of intrinsic tendon cells, indicating that the tendon does not directly contribute to healing. We hypothesize that cytokines produced by infiltrating macrophages are likely to contribute to the formation of a fibrous scar tissue interface rather than a normal insertion site.

Introduction

The histological, biochemical and biomechanical characteristics of ligament-substitution procedures in the knee have been widely investigated. However, the natural history and biology of the tendon–bone interface is still not completely understood. Previous studies demonstrate that tendon-to-bone healing proceeds by formation of a fibrovascular interface tissue between the tendon and bone, followed by bone-ingrowth into the tendon–bone interface and development of collagen fiber continuity between tendon and bone [22], [23]. The cell types that initiate and regulate the process of healing at the tendon-to-bone junction and the temporal sequence of this process are still unknown.

The complex morphology of the normal ligament insertion site is not consistently reformed during healing; rather, a scar interface forms between the tendon and bone. It is our hypothesis that inflammatory cell accumulation following tendon-to-bone repair results in this scar interface. Inflammatory cells produce cytokines (such as transforming growth factor-β) that are known to stimulate fibroblasts and increase production of extracellular matrix [2]. Identification of strategies to improve tendon-to-bone healing will require definition of the molecular signals (patterns of expression of genes for matrix proteins, transcription factors, cytokines, etc.) and cell types at the healing tendon–bone junction. An important step in identifying these molecular signals is to characterize the specific cell types during the healing process.

Several cell types may contribute to healing at the tendon–bone interface. Accumulation of cells in the tendon–bone interface requires either recruitment of new cells from the circulatory system or mitogenesis of resident cells from the surrounding bone marrow, joint cavity, or the tendon graft. The earliest cellular response following surgical implantation of a tendon graft in a bone tunnel most likely involves accumulation of inflammatory cells. It is likely that macrophages play an important role in tendon healing since these cells function to scavenge cellular debris following injury and they can release growth factors that induce extracellular matrix synthesis [13] and stimulate fibroblasts or other stem cells to proliferate [5]. Previous studies of wound healing in other tissues clearly indicate that macrophage accumulation is an early event in the healing process [21]. For example, Wright et al. recently reported that macrophage-lineage cells accumulate in a healing medial collateral ligament injury in a mouse model [28].

The purpose of this study is to test the hypothesis that hematopoietic lineage cells contribute to healing in the early phase of tendon-to-bone healing. We have used a newly-developed rodent model of anterior cruciate ligament (ACL) reconstruction to identify the spatial and temporal pattern of cell proliferation at the healing tendon–bone interface, using immunohistochemical techniques to measure the concentration of neutrophils, recruited macrophages (ED1), resident macrophages (ED2), T-lymphocytes, endothelial cells, mast cells, and pericytes in the early phase of tendon-to-bone healing.

Section snippets

Surgical model

The experimental protocol was approved by the Institutional Animal Care and Use Committee at our institution. Thirty-six Lewis rats (18 males and 18 females) were used and kept in soft bedding cages with free access to food and water. The animals were anesthetized with a mixture of ketamine hydrochloride 80 mg/kg and xylazine 5 mg/kg administered intraperitoneally via a 25-gauge needle. Ampicillin (25 mg/kg subcutaneous) was used for antibiotic prophylaxis. A longitudinal incision was made on the

Observations on the rat ACL reconstruction model

All animals resumed normal cage activity by the first post-operative day, with no evidence of lameness. Careful inspection of the joints at sacrifice demonstrated all grafts to be intact. The articular cartilage surfaces appeared normal, with no evidence of progressive joint degeneration. There was a small amount of thickened synovium around the anterior aspect of the ACL grafts.

Tendon–bone interface

Healing between the tendon and bone occurred by an initial formation of a fibrovascular granulation tissue between

Discussion

Previous studies have shown that healing between a tendon graft and bone occurs by formation of a fibrovascular interface tissue and new woven bone between the bone and tendon [1], [22], [23]. This study has identified the cell types that contribute to the initial formation of this interface tissue. We found that neutrophils, ED1+ and ED2+ macrophages accumulate sequentially following transplantation of a tendon graft in a bone tunnel. These cells are likely to play an important role in the

Acknowledgment

This study was funded by a Pilot and Feasibility Grant (S.A.R.) as part of an N.I.H. Core Centers grant (#1P30AR46121-01). This investigation was conducted in a facility constructed with support from Research Facilities Improvement Program Grant Number C06-RR12538-01 from the National Center for Research Resources, National Institutes of Health.

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