Two Mechanisms of Intrasynovial Tendon Healing

It is now generally believed that intrasynovial flexor tendons heal through two mechanisms—intrinsic and extrinsic. Both intrinsic and extrinsic tendon healing mechanisms come into play in a clinical setting. Intrinsic healing takes place through proliferation of tenocytes and production of extracellular matrix by intrinsic cells. Extrinsic healing is the healing process brought about by growth of tissues or cell seeding from outside the lacerated tendon. Clinically, the participation of intrinsic and extrinsic healing after tendon repair depends on the condition of the tendon and surrounding tissues. Extrinsic cells participate minimally in the healing of clean-cut wounds but can dominate healing when the intrinsic healing is jeopardized by severe trauma to the tendon.

For a typically lacerated tendon, the healing process generally consists of three stages: inflammation, collagen production (or proliferation and repair), and collagen remodeling.

The first stage is manifested with infiltration of inflammatory cells, such as neutrophils. Monocytes and macrophages are recruited to the injury site within the first 24 hours, and phagocytosis of necrotic materials occurs. After the release of vasoactive and chemotactic factors, angiogenesis and proliferation of tenocytes are initiated. Tenocytes then move into the site and start to synthesize collagen III. The inflammation stage usually lasts for a few days, followed by the collagen production stage.

In the collagen production stage, which lasts for 5 to 6 weeks, the tenocytes produce large amounts of collagen and proteoglycans at the repair site. In the early part of this stage, tenocytes proliferate dramatically, but in later weeks, inflammation subsides and cell proliferation is less obvious. Collagen I, which increases healing strength, is produced abundantly in the later period.

At about 6 weeks after injury, the remodeling stage begins. The first part of this stage is consolidation, which lasts from about 6 to 10 weeks after the injury. During this time, synthesis of collagens and glycosaminoglycans (GAGs) decreases, as does cellularity. The tissue becomes more fibrous as a result of increased production of collagen I, and the fibrils become aligned in the direction of mechanical stress. The final maturation stage occurs after 10 weeks, and there is an increase in cross-linking of the collagen fibrils, which makes the tissue stiffer.

Molecular Biology of Tendon Healing

Roles of Individual Growth Factors

A variety of molecules are involved in tendon healing.³⁶ The roles of six growth factors have been studied associated with tendon healing in vivo or tenocyte proliferation in vitro:

1. Transforming growth factor (TGF). This molecule is secreted by all of cell types participating in the healing process and plays a role in wound healing and scar formation. TGF-β has three isoforms: TGF-β1, TGF-β2, and TGF-β3; they play different roles in scar formation. While TGF-β1 and -β2 are known to induce fibrotic changes and scar formation, TGF-β3 may inhibit scar formation.³⁷ Three isoforms are 60% to 80% homologous and are dimers of 12.5-kDa polypeptides cleaved from larger precursors after being secreted from the cells into the ECM. TGF-β is a master mediator in the pathogenesis of fibrosis and scar formation. Regarding specific roles of TGF-β in flexor tendon healing, Chang and colleagues found increased levels of TGF-β1 mRNA in the sheath and the tendon itself after tendon trauma. TGF-β receptors 1, 2, and 3 were upregulated as well. In in vitro studies, TGF-β1 induced collagen I production by tenocytes.¹¹ Chang and colleagues applied TGF-β1 neutralizing antibodies to injured digital flexor tendons, and this therapy increased the range of digital motion of the toes operated upon in a rabbit model.¹³

2. Insulin-like growth factor (IGF). It is a single-chain polypeptide hormone. The primary biological action of IGF-1 is mitogenic activity for a broad range of cells. Extracts from the epitenon and internal compartment of avian flexor tendons were found to contain IGF-1; these extracts could stimulate DNA synthesis by tenocytes.⁸ In in vitro culture of tenocytes, IGF-1 was found to increase cell proliferation as well as stimulate synthesis of DNA, collagen, and proteoglycan.⁹ Both IGF-1 and IGF-2 were shown to promote proliferation of tenocytes in vitro tenocyte culture.⁸^–¹⁰

3. Platelet-derived growth factor (PDGF). PDGF consists of a group of dimers, each of two chains (A and B). Various grouping of these chains form three isoforms (AA, BB, and AB). PDGF acts as a chemoattractant and mitogen for fibroblasts, endothelial cells, and smooth muscle cells. The cell surface contains two kinds of receptors (α and β) for PDGF binding. In fibroblasts, the β receptor is predominant. Only the isoform BB of PDGF binds to the β receptor. Investigations have focused on the roles of PDGF-BB in tendon healing. Exogenous PDGF-BB stimulates collagen, proteoglycan, and DNA synthesis of the tenocytes.¹⁸ Expression of PDGF-BB is increased after tendon injuries in canine and chicken models.^34,³⁷

4. Vascular endothelial growth factor (VEGF). VEGF has five isoforms (i.e., 121, 145, 165, 189, and 206) distinguished by their number of amino acids. Biological effects of these isoforms are similar. VEGF121 and VEGF165 are the predominant isoforms secreted by most cells. VEGFs bind to one of three VEGF receptors (VEGFR 1, 2, and 3) on the cell surface to exert their biologic effects. The primary role of VEGF is to induce angiogenesis, including that in pathological conditions. In a canine model, VEGF mRNA expression was increased in healing flexor tendons.^17,^38,³⁹ VEGF expression may differ between the cells in endotenon and epitenon at the repair site: 67% of the cells in the endotenon at the repair site express VEGF, but 10% of epitenon cells express VEGF.

Direct injection of VEGF increased the healing strength of Achilles tendon in a rat model. However, the effectiveness of direct use of VEGF in synovial tendons has not been tested. We found that AAV2-VEGF treatment significantly increased the healing strength of the chicken flexor tendons at postoperative weeks 3, 4, and 6.

5. Basic fibroblast growth factor (bFGF). bFGF commonly presents as an 18-kDa, single-chain polypeptide of 146 amino acids; other forms of greater molecular weights (22, 22.5, and 24 kDa) also exist. bFGF has a wide range of biological effects, including induction of cell proliferation and migration as well as stimulation of fibroblasts to produce collagens and collagenase. There have been several specific investigations of the roles of bFGF in flexor tendon healing. Duffy and colleagues detected presence of bFGF in normal intrasynovial flexor tendons.¹⁶ Chang and colleagues found that bFGF levels were increased in flexor tendons and sheath during healing from day 1 to week 8.¹² Tang and colleagues observed that exogenous bFGF acts on cultured chicken tenocytes to accelerate cell proliferation and to promote collagen I gene expression.^27,²⁸

Both direct delivery of bFGF and transfer of bFGF cDNA via appropriate vectors to the healing tendons have been tested to enhance tendon healing.^31,⁴⁰^–⁴³ Direct delivery of bFGF was achieved with direct injection of the bFGF,⁴⁰ use of bFGF-coated suture,⁴¹ or bFGF delivery through controlled release system.^42,⁴³ Direct injection of bFGF did not improve the healing strength of the tendon, nor did bFGF delivered through controlled release system.^40,⁴³ After delivery of bFGF to the tendon, biological reactions and increases in cellular proliferation in the healing tendons were observed, but mechanically, the healing strength was not increased.⁴³ Tendon repair with bFGF-coated surgical suture increased the tendon strength at week 3 after surgery, but not at week 6, in a rabbit model.⁴¹ AAV2-bFGF–treated tendons had a significantly greater breaking strength than the nontreatment control at weeks 2, 3, and 4 in a chicken model.³¹

6. Bone morphogenetic proteins (BMPs). BMPs are a group of growth factors originally discovered by their ability to induce the formation of bone and cartilage. BMPs are considered to constitute a group of pivotal morphogenetic signals, orchestrating tissue architecture throughout the body. There are BMPs 1 through 15 in this superfamily. BMP14 is called growth differentiation factor-5 (GDF-5) as well; this factor was particularly studied regarding its role in tendon healing and repair. Hogan and colleagues ⁴⁴ found that growth differentiation factor-5 (GDF-5, or BMP14) regulates ECM gene expression in murine tendon fibroblasts. They isolated mice Achilles tendon fibroblasts and treated them with rGDF-5 (0 to 100 ng/mL) for 0 to 12 days in cell culture. The temporal effect of rGDF-5 on ECM gene expression was analyzed for type 1 collagen and aggrecan expression. They found that expression of extracellular matrix (ECM) genes procollagen IX, aggrecan, matrix metalloproteinase 9, and fibromodulin were upregulated. Proinflammatory reaction genes were downregulated. rGDF-5 led to an increase in total DNA, glycosaminoglycan (GAG), and hydroxyproline (OHP). rGDF-5 treatment showed improved collagen organization over controls.

No studies have directly investigated expression profile of BMPs during intrasynovial tendon healing process. However, studies were performed to determine relation of BMPs with the healing in rat Achilles tendon. Eliasson and colleagues ⁴⁵

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