"Studies of Tendon-to-Bone Healing: Exploring Ways to Improve Graft Fixation Following Anterior Cruciate Ligament Reconstruction"
By Scott A. Rodeo, MD*,
The Laboratory for Soft Tissue Research,
The Hospital for Special Surgery, New York, NY

A Perspective on
"Revision Anterior Cruciate Surgery with Use of
Bone-Patellar Tendon-Bone Autogenous Grafts"
by Frank R. Noyes, MD, and Sue D. Barber-Westin, BS
(JBJS, August 2001)

Reconstruction of the anterior cruciate ligament (ACL) involves use of a tendon graft that is transplanted into bone tunnels at the femoral and tibial insertion sites. Understanding the basic mechanisms of tendon-to-bone healing is important since the graft attachment site is the "weak link" in the early healing period, necessitating a delay in return to function. Furthermore, healing of the tendon graft transplanted in a bone tunnel may be impaired because of graft-tunnel motion or graft-tunnel mismatch, as enlarged tunnels may be encountered in revision reconstruction. Furthermore, widening of the bone tunnel, presumably due to bone resorption around the tunnel, is sometimes observed and may impair graft incorporation¹. Healing of the tendinous portion of the graft is important even when bone-tendon-bone (BTB) grafts are used, because with current endoscopic techniques the tendinous portion of the graft usually extends into the upper part of the tibial tunnel. Since the most important site for secure healing may be at the tunnel opening into the joint ("aperture fixation"), it is obvious that even BTB grafts require tendon-to-bone healing and not just bone-to-bone healing².

The ideal ACL graft would possess microscopic structure and biomechanical characteristics identical to those of the native ACL, including a normal ligament insertion. The normal insertion site is a highly specialized zone of tissue that functions to transmit stress from hard tissue to soft tissue. The native ACL attaches to the bone surface via a direct type of insertion, which contains four distinct histologic zones: 1) ligament, 2) uncalcified fibrocartilage, 3) calcified fibrocartilage, and 4) bone (Fig. 1). There is a tidemark between the zones of calcified and uncalcified cartilage, which represents the mineralization front³. In contrast, tendon grafts are fixed to bone with use of bone tunnels. There are no sites in humans where a tendon goes into a bone tunnel, and thus there is no native situation analogous to a tunnel-graft. On the basis of normal ACL structure and the known function of the insertion site, the ideal ACL graft would attach broadly to the surface of the bone at the femoral and tibial attachment sites by means of an intermediate zone of fibrocartilage.

Fig. 1. The normal direct insertion site contains four distinct histologic zones: tendon (T); unmineralized fibrocartilage (UFC); mineralized fibrocartilage (MFC); and bone (B). The tidemark, representing the mineralization front, is located between the zones of unmineralized fibrocartilage and mineralized fibrocartilage.

Animal studies have demonstrated various mechanisms of healing of a tendon graft in a bone tunnel. Some studies have revealed the formation of a zone of fibrocartilage intermediate between tendon and bone, while in other models graft-healing proceeds by means of fibrous tissue at the tendon-bone interface^4-10. These disparities may be due to differences in the species studied, graft location (intra-articular versus extra-articular), and mechanical environment. There are many fundamental questions about the mechanism of tendon-to-bone healing that remain unanswered, such as: 1) What is the source of the cells that participate in early healing? 2) What is the temporal and spatial pattern of relevant gene expression? 3) What chemical signals (such as cytokines) regulate the healing process? and 4) Is normal attachment strength ever re-established?

We have used a rabbit model of ACL reconstruction in which a semitendinosus tendon graft is transplanted into femoral and tibial bone tunnels to replace the ACL. The graft is attached to the periosteum adjacent to the bone tunnels¹⁰. In this model, which essentially duplicates the procedure used in humans (Fig. 2), we have found that healing begins with proliferation of fibrovascular tissue in the interface between tendon and bone¹⁰. We have used bromodeoxyuridine (BrdU) to label proliferating cells and have found that marrow cells from the surrounding tunnel initiate the healing process. Use of immunohistochemical techniques has demonstrated that many of the early cells in the tendon-bone interface stain with phenotypic markers for macrophage-monocytes (CD-68) and leukocytes (CD-45). These cells are present by 3-7 days following tendon transplantation in our rabbit model⁹. It is likely that there are other types of marrow-derived pluripotential stem cells in the tendon-bone interface that contribute to the healing process. Although occasional proliferation of cells is noted along the edge of the tendon, it appears that the tendon cells do not play an important role in the healing process. The interface cells begin to infiltrate into the outer tendon by 14 days and may contribute to the eventual cellular repopulation of the tendon graft.

Fig. 2. The semitendinosus tendon graft is placed into femoral and tibial drill tunnels to replace the anterior cruciate ligament.

Healing progresses through formation of new matrix in the tendon-bone interface. Fibrous tissue is deposited in the interface in the first 7 days, followed by proliferation of new bone trabeculae along the edge of the tunnel. In some areas there is direct apposition of new bone trabeculae and the fibrous interface, while in other areas we have observed formation of a cartilaginous interface between tendon and bone. New bone and cartilage grow into the fibrous interface, which ultimately results in the attachment of the tendon graft and bone through the re-establishment of collagen fiber continuity. It is not definitively established whether a zone of fibrocartilage will persist between the tendon and bone (as in a normal, direct type of insertion) or if the cartilage that is observed is part of a process of endochondral bone formation. We have not observed formation of a distinct tidemark.

We have noted variability in the type and amount of new tissue formation between the tendon graft and bone tunnel within a single specimen. There is sometimes vigorous tissue formation on one side of the tunnel with minimal tissue formation on the opposite side of the tunnel. This variability may be due to differences in the local mechanical environment. For example, the graft may bend as it enters the tunnel, resulting in compression between tendon and bone on one side of the tunnel and shear forces on the opposite side of the tunnel. Synovial fluid influx may also impede healing in some areas by preventing formation of a fibrin clot in the early healing period. It seems likely that there would be similar variability in healing in humans. Even in areas where fibrocartilage forms in the tendon-bone interface, the morphology differs from that of a normal, direct insertion. Important components of the native insertion site are not re-established, including a distinct tidemark, the zone of calcified cartilage, columnar organization of the chondrocytes, and the consistent, firm collagen fiber anchoring tendon to bone¹⁰.

The little clinical information that is available about tendon-healing in a bone tunnel involves biopsies taken during revision surgery. Specimens from two patients examined by the author demonstrated an interface of fibrous tissue between the tendon graft and the bone. It is not known if a direct type of insertion (with a cartilaginous zone between tendon and bone) would provide superior anchorage.

On the basis of our finding that tendon-to-bone healing proceeds by bone ingrowth into the fibrous tissue interface, we have used exogenous osteoinductive agents in an attempt to augment this healing. We have demonstrated improved healing by applying a mixture of bone-derived growth factors (Ne-Osteo; Sulzer Biologics, Denver, Colorado) at the tendon-bone interface in our rabbit ACL reconstruction model¹⁰. Histologic analysis showed increased bone and cartilage formation around the tendon graft, and biomechanical testing showed a significant increase in attachment strength compared with that in controls at 2, 4, and 8 weeks following tendon transplantation (p = 0.04, p = 0.01, and p < 0.001, for 2, 4, and 8 week time-points, respectively). We have also found improved bone formation around a tendon graft using exogenous recombinant human bone morphogenetic protein-2 (rhBMP-2; Genetics Institute, Cambridge, Massachusetts) in an extra-articular bone tunnel in a dog model¹¹. In that study there was a significant increase in attachment strength compared with that in controls only at 2 weeks (p = 0.014). These agents are also similarly effective in large animals; Nicklin et al. demonstrated that exogenous osteogenic protein-1 (OP-1) results in improved bone formation at the tendon-bone interface in a sheep model of ACL reconstruction¹². These studies demonstrate the potential of biologic agents to improve healing in our patients.

Our studies also provide some insight into the possible mechanism of tunnel widening. The normal healing process involves osteoclastic resorption along the edge of the tunnel in the early time-points following tendon transplantation. We have used tartrate-resistant acid phosphatase histochemistry to localize osteoclasts at the edge of the bone tunnel. We also reported that application of a high dose of rhBMP-2 resulted in excessive osteoclast-mediated bone resorption by two weeks following tendon transplantation in our dog model¹¹. The radiographic appearance was similar to that seen in patients with tunnel widening. This was followed subsequently by vigorous new bone formation in the tendon-bone interface. It is possible that alterations in cytokine expression or in the balance of specific cytokines result in excessive bone resorption. The exact mechanisms and implications of tunnel widening in patients are currently unknown.

Further knowledge of the basic biology of tendon-to-bone healing will also help us to gain a better understanding of the potential differences in graft-fixation devices. A number of implants are available for fixation of tendon grafts into a bone tunnel, including devices that directly fix a graft in the tunnel (such as an interference screw), devices that suspend a graft within the tunnel (such as the Bone Mulch Screw; Arthrotek, Warsaw, Indiana), and devices that fix the graft outside the tunnel (such as Endobutton). These devices are likely to result in substantial differences in the mechanical environment, which in turn are likely to affect the healing process. For example, it is established that there is higher strain in a tendon graft that is attached outside of the tunnel than one secured at the normal anatomic attachment site of the native ligament (aperture fixation)². Furthermore, absorbable devices in the bone tunnel (such as bioabsorbable interference screws) may result in changes in the chemical environment during implant resorption, which may also affect healing.

In conclusion, studies of the basic mechanisms of tendon-to-bone healing may lead to new methods of treatment that improve healing. These methods could apply to any situation in which a tendon graft is placed into bone tunnels, such as anterior or posterior cruciate ligament reconstruction in the knee, ankle ligament reconstruction, or ulnar collateral ligament reconstruction in the elbow. Knowledge of gene expression at healing tendon insertion sites will suggest ways to manipulate the proper chemical and molecular signals in order to improve healing. This information will also be applicable to the attachment of tissue-engineered neoligament grafts.

*The author did not receive grants or outside funding in support of his research or preparation of this manuscript. He received payments or other benefits or a commitment or agreement to provide such benefits from commercial entities (Genetics Institute and Sulzer Biologics). In addition, commercial entities (Genetics Institute and Sulzer Biologics) paid or directed, or agreed to pay or direct, benefits to a research fund, foundation, educational institution, or other charitable or nonprofit organization with which the author is affiliated or associated.

References

1. Fahey M, Indelicato PA. Bone tunnel enlargement after anterior cruciate ligament replacement. Am J Sports Med. 1994;22:410-4.
2. Hoher J, Scheffler SU, Withrow JD, Livesay GA, Debski RE, Fu FH, Woo SL. Mechanical behavior of two hamstring graft constructs for reconstruction of the anterior cruciate ligament. J Orthop Res. 2000;18:456-61.
3. Cooper RR, Misol S. Tendon and ligament insertion. A light and electron microscopic study. J Bone Joint Surg Am. 1970;52:1-20.
4. Grana WA, Egle DM, Mahnken R, Goodhart CW. An analysis of autograft fixation after anterior cruciate ligament reconstruction in a rabbit model. Am J Sports Med. 1994;22:344-51.
5. Park MJ, Seong SC, Lee MC. A comparative study on healing of bone-tendon autograft and bone-tendon-bone autograft using patellar tendon in rabbits. Orthop Trans. 1998;23:610.
6. Schiavone Panni A, Fabbriciani C, Delcogliano A, Franzese S. Bone-ligament interaction in patellar tendon reconstruction of the ACL. Knee Surg Sports Traumatol Arthrosc. 1993;1:4-8.
7. Panni AS, Milano G, Lucania L, Fabbriciani C. Graft healing after anterior cruciate ligament reconstruction in rabbits. Clin Orthop. 1997;343:203-12.
8. Shino K, Kawasaki T, Hirose H, Gotoh I, Inoue M, Ono K. Replacement of the anterior cruciate ligament by an allogeneic tendon graft. An experimental study in the dog. J Bone Joint Surg Br. 1984;66:672-81.
9. Kim HJ, Hatch J, Abbot A, Ying L, McCarthy D, Rodeo SA. Identification of the cells that participate in early tendon-to-bone healing. Orthop Trans. 2001;26:742.
10. Anderson K, Seneviratne AM, Izawa K, Atkinson BL, Potter HG, Rodeo SA. Augmentation of tendon healing in an intra-articular bone tunnel using a bone growth factor. Am J Sports Med. 2001; in press.
11. Rodeo SA, Suzuki K, Deng XH, Wozney J, Warren RF. Use of recombinant human bone morphogenetic protein-2 to enhance tendon healing in a bone tunnel. Am J Sports Med. 1999;27:476-88.
12. Nicklin S, Morris H, Yu Y, Harrison J, Walsh WR. OP-1 augmentation of tendon-bone healing in an ovine ACL reconstruction. Orthop Trans. 2000;25:155.