Elsevier

Biomaterials

Volume 28, Issue 2, January 2007, Pages 187-202
Biomaterials

Review
Techniques for biological characterization of tissue-engineered tendon and ligament

https://doi.org/10.1016/j.biomaterials.2006.08.040Get rights and content

Abstract

Injuries to tendons and ligaments are prevalent and result in a significant decrease in quality of patient life. Tissue-engineering strategies hold promise as alternatives to current treatments for these injuries, which often fail to fully restore proper joint biomechanics and produce significant donor site morbidity. Commonly, tissue engineering involves the use of a three-dimensional scaffold seeded with cells that can be directed to form tendon/ligament tissue. When determining the success of such approaches, the viability and proliferation of the cells in the construct, as well as extracellular matrix production and structure should be taken into account. Histology and histochemistry, microscopy, colorimetric assays, and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) are techniques that are employed to assess these biological characteristics. This review provides an overview of each of these methods, including specific examples of how they have been used in evaluation of tissue-engineered tendon and ligament tissue. Basic physical principles underlying each method and advantages and disadvantages of the various techniques are summarized.

Introduction

Over 800,000 people seek medical attention each year for injuries to ligaments, tendons, or the joint capsule [1]. Ruptures and other damage to tendons and ligaments can cause great pain and decrease the functionality of the joint complex. There are numerous areas throughout the body where tendons and ligaments experience such injuries. These include the patellar tendon [2], the anterior cruciate ligament [3], the posterior cruciate ligament [4], and the medial collateral ligament [5] in the knee; the Achilles tendon [6] at the heel; the anterior talofibular ligament [7] and the calcaneofibular ligament [8] in the ankle; the radial ulnohumeral ligament [9] and the lateral ulnar collateral ligament [10] in the elbow; the digital flexor tendon [3], [6] and the ulnar collateral ligament [11] in the hand; the scapholunate ligament [12] in the wrist; the rotator cuff tendons [3], the acromioclavicular ligament [13], the coracoclavicular ligament [14], and the coracohumeral ligament [15] at the shoulder; and the gluteus medius tendon [16] at the hip.

Often, natural healing of these injuries is insufficient since many tendons and ligaments possess a limited capacity to regenerate [6], [17]. While certain tendons and ligaments can be repaired by suturing the injured tissue back together, some heal poorly in response to this type of surgery, so the use of grafts is required [18]. Unfortunately, finding suitable graft material can be problematic. Autografts from the patient may result in donor site morbidity, while allografts from cadavers may cause a harmful response from the immune system and are limited in supply [19]. In both cases, the graft often does not match the strength of undamaged tissue [19].

Such shortcomings in repair of tendons and ligaments may be overcome through tissue-engineering approaches. Tissue-engineering strategies involve the use of a biomaterial carrier, cells, and/or bioactive factors to promote tissue regeneration via natural processes in the body [20]. One common tissue-engineering approach involves using a three-dimensional (3D) scaffold to direct pre-seeded cells to create viable tendon/ligament tissue [21], [22], [23], [24], [25]. In order to determine the relative success between different tissue-engineering techniques, specific outcome measures (success criteria) are required. Biological assessment, in conjunction with other tests, is a crucial part of the optimization and final clinical application of tissue-engineered tendons and ligaments, and, thus, is the subject of this review. A variety of techniques exist to characterize biological components of tendon and ligament tissue, but only a subset of these methods has been employed to evaluate tissue-engineered tendons and ligaments. Therefore, this review focuses on those most commonly used to assess biological parameters of tissue-engineered tendon/ligament constructs.

Section snippets

Function and structure of tendon and ligament tissue

Biological evaluation is carried out on tissue-engineered constructs to determine to what extent the construct tissue replicates the structure and chemical composition of native tendon/ligament tissue. Thus, a brief review of the structure and function of tendons and ligaments is included in this section.

Tissue-engineering approaches

There are many approaches that exist for tissue engineering of tendons and ligaments. Commonly, a 3D scaffold housing a specific cell type that can be directed to form tendon/ligament tissue is employed. 3D culture offers the advantage of more closely recreating the spatial organization of native tissue than two-dimensional culture.

There are many considerations that should be taken into account when engineering tendon or ligament tissue [1]. The scaffold should encourage cellular recruitment

Types of biological characterization techniques

A variety of techniques are available to examine construct characteristics, including histology, microscopy, colorimetry, and reverse transcriptase-polymerase chain reaction (RT-PCR). The basic theory behind each method is explained in this section, while examples of how these techniques are used to assess tissue-engineered tendon and ligament are found in Section 5.

Examples of biological assessment of tendon and ligament constructs

When determining the success of tendon and ligament tissue-engineering techniques, the viability and proliferation of the cells in the construct, the amount and type of ECM production, and the spatial organization of the ECM components should be taken into account. Since the cells are the source of ECM and renewal of the tissue, their viability is crucial to the success of the tissue-engineering technique. The ECM molecules that the cells secrete are the largest component of tendon and ligament

Conclusions

Tissue engineering provides the possibility for repair and regeneration of tendon and ligament injuries. In order to determine the success of tissue-engineering methods, it is important to assess biological characteristics of the newly formed tissue, including cellularity and ECM production and structure. While established techniques, such as those described here, have provided valuable basic insights, each has its own associated limitations. More sophisticated analysis of tissue-engineered

Acknowledgments

This work was supported in part by the Georgia Tech/Emory Center for the Engineering of Living Tissues, a National Science Foundation Research Center (EEC-9731643).

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