Dense Connective Tissues Research Articles

Optical clearing in collagen- and proteoglycan-rich osteochondral tissues

Recent developments in optical clearing and microscopy technology have enabled the imaging of intact tissues at the millimeter scale to characterize cells via fluorescence labeling. While these techniques have facilitated the three-dimensional (3D) cellular characterization within brain and heart, study of dense connective tissues of the musculoskeletal system have been largely unexplored. Here, we quantify how optical clearing impacted the cell and tissue morphology of collagen-, proteoglycan-, and mineral-rich cartilage and bone from the articulating knee joint. Water-based fructose solutions were used for optical clearing of bovine osteochondral tissues, followed by imaging with transmission and confocal microscopy. To confirm preservation of tissue structure during the clearing process, samples were mechanically tested in unconfined compression and visualized by cryo-SEM. Optical clearing enhanced light transmission through cartilage, but not subchondral bone regions. Fluorescent staining and immunolabeling was preserved through sample preparations, enabling imaging to cartilage depths five times deeper than previously reported, limited only by the working distance of the microscope objective. Chondrocyte volume remained unchanged in response to, and upon the reversal, of clearing. Equilibrium modulus increased in cleared samples, and was attributed to exchange of interstitial fluid with the more viscous fructose solution, but returned to control levels upon unclearing. In addition, cryo-SEM-based analysis of cartilage showed no ultrastructural changes. We anticipate large-scale microscopy of diverse connective tissues will enable the study of intact, 3D interfaces (e.g., osteochondral) and cellular connectivity as a function of development, disease, and regeneration, which have been previously hindered by specimen opacity.

Repair of dense connective tissues via biomaterial-mediated matrix reprogramming of the wound interface

Repair of dense connective tissues in adults is limited by their intrinsic hypocellularity and is exacerbated by a dense extracellular matrix (ECM) that impedes cellular migration to and local proliferation at the wound site. Conversely, healing in fetal tissues occurs due in part to an environment conducive to cell mobility and division. Here, we investigated whether the application of a degradative enzyme, collagenase, could reprogram the adult wound margin to a more fetal-like state, and thus abrogate the biophysical impediments that hinder migration and proliferation. We tested this concept using the knee meniscus, a commonly injured structure for which few regenerative approaches exist. To focus delivery and degradation to the wound interface, we developed a system in which collagenase was stored inside poly(ethylene oxide) (PEO) electrospun nanofibers and released upon hydration. Through a series of in vitro and in vivo studies, our findings show that partial digestion of the wound interface improves repair by creating a more compliant and porous microenvironment that expedites cell migration to and/or proliferation at the wound margin. This innovative approach of targeted manipulation of the wound interface, focused on removing the naturally occurring barriers to adult tissue repair, may find widespread application in the treatment of injuries to a variety of dense connective tissues.

Collagen XII: Protecting bone and muscle integrity by organizing collagen fibrils

Collagen XII, largest member of the fibril-associated collagens with interrupted triple helix (FACIT) family, assembles from three identical α-chains encoded by the COL12A1 gene. The molecule consists of three threadlike N-terminal noncollagenous NC3 domains, joined by disulfide bonds and a short interrupted collagen triple helix toward the C-terminus. Splice variants differ considerably in size and properties: “small” collagen XIIB (220kDa subunit) is similar to collagen XIV, whereas collagen XIIA (350kDa) has a much larger NC3 domain carrying glycosaminoglycan chains. Collagen XII binds to collagen I-containing fibrils via its collagenous domain, whereas its large noncollagenous arms interact with other matrix proteins such as tenascin-X. In dense connective tissues and bone, collagen XII is thought to regulate organization and mechanical properties of collagen fibril bundles. Accordingly, recent findings show that collagen XII mutations cause Ehlers–Danlos/myopathy overlap syndrome associated with skeletal abnormalities and muscle weakness in mice and humans.

Gross, Histological and Ultrastructural Features of the Bulbourethral Gland in the Greater Cane Rat (Thryonomys swinderianus)

The present study examines the structure and ultrastructure of the bulbourethral glands in 10 sexually matured male greater cane rats raised in captivity. Following anaesthesia, the rats were perfusion-fixed transcardially and the bulbourethral glands dissected out. Upon morphologic and morphometric analysis, the Cowper's glands were observed to have an average volume of 0.24±0.08 ml, a diameter of 6.3±0.6 mm and weighs 0.199±0.06 g. The paired, gourd-shaped tubuloalveolar glands were surrounded by dense connective tissues and separated into lobules by capsular septae. Each lobule consists of endpiece/secretory units and excretory ducts lined by simple glandular epithelium and pseudo-stratified epithelium, respectively. The round end pieces consisted of 8-10 pyramidal to columnar epithelial cells with flattened, basally located nuclei and granule-filled cytoplasm that bounded a narrow glandular lumen. The striking ultrastructural features of these secretory cells were the presence of some granules with uniform electron density and those with regions of lesser density as well as the absence of secretory vacuoles. Another unique characteristic of these secretory granules is the presence of electron dense strands radiating from their surfaces. The apical surfaces of the cells were also studded with abundant microvilli. From the findings, the structure of bulbourethral glands in the greater cane rat shows more resemblances to that of humans than to its rodent phylogeny. These findings serve as additional knowledge in the structural interpretation of the bulbourethral gland and its secretory products.

Clinical and radiographic findings and usefulness of computed tomographic assessment in two children with regional odontodysplasia.

Regional odontodysplasia is a rare, severe, and nonhereditary developmental disorder in tooth formation and involves epithelial and mesenchymal-derived dental tissue. On radiographs, affected teeth have an abnormal morphology, a hypoplastic crown, and only a faint outline of hard tissue, a condition termed “ghost teeth.” We report clinical and radiographic findings from two children with regional odontodysplasia. Using computed tomography (CT), we calculated attenuation coefficients (i.e., Hounsfield units) for affected teeth and assessed the condition of dental follicles. To measure density, regions of interest were delimited and CT values were calculated. In our two patients, the CT values for enamel were lower in affected teeth than in sound teeth, while CT values for dentin were similar for affected and sound teeth. The average CT value for dental follicles in affected teeth was about 65 to 120, which suggests that dense fibrous connective tissues or hard tissue-like structures might be present in dental follicles. Analysis of CT values may be quite useful in the diagnosis and treatment of regional odontodysplasia.

INTRAHEPATIC CHOLANGIOCARCINOMA IN A CAPTIVE MEERKAT (SURICATA SURICATTA)

A 9-yr-old male meerkat (Suricata suricatta) living in captivity, with a history of anorexia, lethargy, and weight loss, was examined postmortem. Physical examination revealed poor body condition, dehydration, and icteric mucous membranes. Macroscopically, white to yellowish, multinodulated masses were found protruding from the liver. These multinodular masses were also observed in all lobes of the lungs and the mediastinal lymph nodes. Microscopic examination revealed tumors with well-circumscribed, atypical proliferating cuboidal to columnar bile duct epithelial layers arranged in solid sheets and papillary patterns. The neoplastic masses were separated by dense fibrous connective tissues and invaded the normal parenchyma. Periodic acid-Schiff-positive material was occasionally found within the lumen of tubuloacinar structures. Immunohistochemical labeling revealed that neoplastic cells were intensely positive for pan-cytokeratin, but negative for vimentin. Based on the macroscopic and microscopic findings, intrahepatic cholangiocarcinoma was diagnosed. This is the first report describing cholangiocarcinoma in a meerkat.

Tendon and ligament regeneration and repair: Clinical relevance and developmental paradigm

As dense connective tissues connecting bone to muscle and bone to bone, respectively, tendon and ligament (T/L) arise from the somitic mesoderm, originating in a recently discovered somitic compartment, the syndetome. Inductive signals from the adjacent sclerotome and myotome upregulate expression of Scleraxis, a key transcription factor for tenogenic and ligamentogenic differentiation. Understanding T/L development is critical to establishing a knowledge base for improving the healing and repair of T/L injuries, a high-burden disease due to the intrinsically poor natural healing response. Current treatment of the three most common tendon injuries-tearing of the rotator cuff of the shoulder, flexor tendon of the hand, and Achilles tendon-include mostly surgical repair and/or conservative approaches, including biophysical modalities such as rehabilitation and cryotherapy. Unfortunately, the fibrovascular scar formed during healing possesses inferior mechanical and biochemical properties, resulting in compromised tissue functionality. Regenerative approaches have sought to augment the injured tissue with cells, scaffolds, bioactive agents, and mechanical stimulation to improve the natural healing response. The key challenges in restoring full T/L function following injury include optimal combination of these biological agents as well as their delivery to the injury site. A greater understanding of the molecular mechanisms involved in T/L development and natural healing, coupled with the capability of producing complex biomaterials to deliver multiple biofactors with high spatiotemporal resolution and specificity, should lead to regenerative procedures that more closely recapitulate T/L morphogenesis, thereby offering future patients the prospect of T/L regeneration, as opposed to simple tissue repair.

Tenomodulin Expression in the Periodontal Ligament Enhances Cellular Adhesion

Tenomodulin (Tnmd) is a type II transmembrane protein characteristically expressed in dense connective tissues such as tendons and ligaments. Its expression in the periodontal ligament (PDL) has also been demonstrated, though the timing and function remain unclear. We investigated the expression of Tnmd during murine tooth eruption and explored its biological functions in vitro. Tnmd expression was related to the time of eruption when occlusal force was transferred to the teeth and surrounding tissues. Tnmd overexpression enhanced cell adhesion in NIH3T3 and human PDL cells. In addition, Tnmd-knockout fibroblasts showed decreased cell adhesion. In the extracellular portions of Tnmd, the BRICHOS domain or CS region was found to be responsible for Tnmd-mediated enhancement of cell adhesion. These results suggest that Tnmd acts on the maturation or maintenance of the PDL by positively regulating cell adhesion via its BRICHOS domain.

Controlled delivery of mesenchymal stem cells and growth factors using a nanofiber scaffold for tendon repair

Outcomes after tendon repair are often unsatisfactory, despite improvements in surgical techniques and rehabilitation methods. Recent studies aimed at enhancing repair have targeted the paucicellular nature of tendon for enhancing repair; however, most approaches for delivering growth factors and cells have not been designed for dense connective tissues such as tendon. Therefore, we developed a scaffold capable of delivering growth factors and cells in a surgically manageable form for tendon repair. Platelet-derived growth factor BB (PDGF-BB), along with adipose-derived mesenchymal stem cells (ASCs), were incorporated into a heparin/fibrin-based delivery system (HBDS). This hydrogel was then layered with an electrospun nanofiber poly(lactic-co-glycolic acid) (PLGA) backbone. The HBDS allowed for the concurrent delivery of PDGF-BB and ASCs in a controlled manner, while the PLGA backbone provided structural integrity for surgical handling and tendon implantation. In vitro studies verified that the cells remained viable, and that sustained growth factor release was achieved. In vivo studies in a large animal tendon model verified that the approach was clinically relevant, and that the cells remained viable in the tendon repair environment. Only a mild immunoresponse was seen at dissection, histologically, and at the mRNA level; fluorescently labeled ASCs and the scaffold were found at the repair site 9days post-operatively; and increased total DNA was observed in ASC-treated tendons. The novel layered scaffold has the potential for improving tendon healing due to its ability to deliver both cells and growth factors simultaneously in a surgically convenient manner.

Biomaterial-mediated delivery of degradative enzymes to improve meniscus integration and repair

Endogenous repair of fibrous connective tissues is limited, and there exist few successful strategies to improve healing after injury. As such, new methods that advance repair by promoting cell growth, extracellular matrix (ECM) production, and tissue integration would represent a marked clinical advance. Using the meniscus as a test platform, we sought to develop an enzyme-releasing scaffold that enhances integrative repair. We hypothesized that the high ECM density and low cellularity of native tissue present physical and biological barriers to endogenous healing, and that localized collagenase treatment might expedite cell migration to the wound edge and tissue remodeling. To test this hypothesis, we fabricated a delivery system in which collagenase was stored inside electrospun poly(ethylene oxide) (PEO) nanofibers and released upon hydration. In vitro results showed that partial digestion of the wound interface improved repair by creating a microenvironment that facilitated cell migration, proliferation and matrix deposition. Specifically, treatment with high-dose collagenase led to a 2-fold increase in cell density at the wound margin and a 2-fold increase in integrative tissue compared to untreated controls at 4weeks (P⩽0.05). Furthermore, when composite scaffolds containing both collagenase-releasing and structural fiber fractions were placed inside meniscal tears in vitro, enzyme release acted locally and resulted in a positive cellular response similar to that of global treatment with aqueous collagenase. This innovative approach to targeted enzyme delivery may aid the many patients that exhibit meniscal tears by promoting integration of the defect, thereby circumventing the pathologic consequences of partial meniscus removal, and may find widespread application in the treatment of injuries to a variety of dense connective tissues.

Orthotic Considerations for Dense Connective Tissue and Articular Cartilage—The Need for Optimal Movement and Stress

Orthotic intervention is an essential component of hand rehabilitation, addressing biological factors that affect activity and participation. Functional, pain-free joint mobility requires skeletal stability, healthy articular cartilage, and appropriate extensibility of periarticular dense connective tissues (DCTs). This article addresses basic science underlying clinical reasoning when considering orthoses to maintain or restore structural integrity, mobility and function of DCT structures, and articular cartilage. However, these tissues often have different and sometimes conflicting requirements for the maintenance and restoration of integrity and health. The duration of immobilization, especially at end range, should be carefully considered, as it impairs nutrition of tissues and adversely compresses articular cartilage, causing injury that may not be reversible. Immobilization also reduces extensibility of DCT. Thus, an intermittent orthotic wearing schedule is suggested, allowing movement wherever possible to promote tissue health. To optimize benefits and minimize harmful effects of orthotic intervention, further research on physiological responses of human tissues to immobilization and tension is needed.

Fabrication and evaluation of biomimetic-synthetic nanofibrous composites for soft tissue regeneration

Electrospun scaffolds hold promise for the regeneration of dense connective tissues, given their nanoscale topographies, provision of directional cues for infiltrating cells and versatile composition. Synthetic slow-degrading scaffolds provide long-term mechanical support and nanoscale instructional cues; however, these scaffolds suffer from a poor infiltration rate. Alternatively, nanofibrous constructs formed from natural biomimetic materials (such as collagen) rapidly infiltrate but provide little mechanical support. To take advantage of the positive features of these constructs, we have developed a composite scaffold consisting in both a biomimetic fiber fraction (i.e., Type I collagen nanofibers) together with a traditional synthetic (i.e., poly-[ε-caprolactone], PCL) fiber fraction. We hypothesize that inclusion of biomimetic elements will improve initial cell adhesion and eventual scaffold infiltration, whereas the synthetic elements will provide controlled and long-term mechanical support. We have developed a method of forming and crosslinking collagen nanofibers by using the natural crosslinking agent genipin (GP). Further, we have formed composites from collagen and PCL and evaluated the long-term performance of these scaffolds when seeded with mesenchymal stem cells. Our results demonstrate that GP crosslinking is cytocompatible and generates stable nanofibrous type I collagen constructs. Composites with varying fractions of the biomimetic and synthetic fiber families are formed and retain their collagen fiber fractions during in vitro culture. However, at the maximum collagen fiber fractions (20%), cell ingress is limited compared with pure PCL scaffolds. These results provide a new foundation for the development and optimization of biomimetic/synthetic nanofibrous composites for in vivo tissue engineering.

Tissue Engineering Strategies in Ligament Regeneration

Ligaments are dense fibrous connective tissues that connect bones to other bones and their injuries are frequently encountered in the clinic. The current clinical approaches in ligament repair and regeneration are limited to autografts, as the gold standard, and allografts. Both of these techniques have their own drawbacks that limit the success in clinical setting; therefore, new strategies are being developed in order to be able to solve the current problems of ligament grafting. Tissue engineering is a novel promising technique that aims to solve these problems, by producing viable artificial ligament substitutes in the laboratory conditions with the potential of transplantation to the patients with a high success rate. Direct cell and/or growth factor injection to the defect site is another current approach aiming to enhance the repair process of the native tissue. This review summarizes the current approaches in ligament tissue engineering strategies including the use of scaffolds, their modification techniques, as well as the use of bioreactors to achieve enhanced regeneration rates, while also discussing the advances in growth factor and cell therapy applications towards obtaining enhanced ligament regeneration.

Strain hardening of fascia: Static stretching of dense fibrous connective tissues can induce a temporary stiffness increase accompanied by enhanced matrix hydration

This study examined a potential cellular basis for strain hardening of fascial tissues: an increase in stiffness induced by stretch and subsequent rest. Mice lumbodorsal fascia were isometrically stretched for 15 min followed by 30 min rest (n=16). An increase in stiffness was observed in the majority of samples, including the nonviable control samples. Investigations with porcine lumbar fascia explored hydration changes as an explanation (n=24). Subject to similar loading procedures, tissues showed decreases in fluid content immediately post-stretch and increases during rest phases. When allowed sufficient resting time, a super-compensation phenomenon was observed, characterised by matrix hydration higher than initial levels and increases in tissue stiffness. Therefore, fascial strain hardening does not seem to rely on cellular contraction, but rather on this super-compensation. Given a comparable occurrence of this behaviour in vivo, clinical application of routines for injury prevention merit exploration.

Conversion of Human Bone Marrow-Derived Mesenchymal Stem Cells into Tendon Progenitor Cells by Ectopic Expression of Scleraxis

Tendons and ligaments (T/L) are dense connective tissues of mesodermal origin. During embryonic development, the tendon-specific cells descend from a sub-set of mesenchymal progenitors condensed in the syndetome, a dorsolateral domain of the sclerotome. These cells are defined by the expression of the transcription factor scleraxis (Scx), which regulates tendon formation and several other characteristic genes, such as collagen type I, decorin, fibromodulin, and tenomodulin (Tnmd). In contrast to other mesenchymal progenitors, the genealogy and biology of the tenogenic lineage is not yet fully understood due to the lack of simple and efficient protocols enabling generation of progenitors in vitro. Here, we investigated whether the expression of Scx can lead to the direct commitment of mesenchymal stem cells (MSCs) into tendon progenitors. First, MSC derived from human bone marrow (hMSC) were lentivirally transduced with FLAG-Scx cDNA to establish 2 clonal cell lines, hMSC-Scx and hMSC-Mock. Subsequent to Scx transduction, hMSC underwent cell morphology change and had significantly reduced proliferation and clonogenicity. Gene expression analysis demonstrated that collagen type I and several T/L-related proteoglycans were upregulated in hMSC-Scx cells. When stimulated toward 3 different mesenchymal lineages, hMSC-Scx cells failed to differentiate into chondrocytes and osteoblasts, whereas adipogenic differentiation still occurred. Lastly, we detected a remarkable upregulation of the T/L differentiation gene Tnmd in hMSC-Scx. From these results, we conclude that Scx delivery results in the direct programming of hMSC into tendon progenitors and that the newly generated hMSC-Scx cell line can be a powerful and useful tool in T/L research.

Ontogeny of the parietal frill of Triceratops: A preliminary histological analysis

The parietal frill of Triceratops, one of the largest cranial ornamental features known, undergoes extraordinary morphological changes late in ontogeny — progressing from a large, thickened, solid frill to a substantially larger, thin, fenestrated frill. To understand how this structure changed so dramatically we undertook a histological examination of the caudal end of an ontogenetic series of Triceratops parietals. Investigation revealed a histological progression that involved an initial period of non-pathologic hyperostosis, followed by a phase of external (dorsal and ventral sides of the parietal) resorption and border extension, with a conclusive stage hypothesized to be dense fibrous connective tissues mineralized through the process of metaplasia. These fibrous tissues form the caudal end of the mature parietal as well as the epiparietals that fuse to the caudal border of the frill late in ontogeny. Continued resorption near the central parietal regions of the left and right lateral portions of the parietal eventually results in a pair of large circular fenestrae. This progression is somewhat similar to the parietal ontogeny of Centrosaurus.

Controlled collagen assembly to build dense tissue-like materials for tissue engineering

Intermediate states of matter, in the form of liquid crystals, are indirectly evidenced in the body. Hence concepts developed by the soft matter community have allowed the highlighting of original morphogenetic pathways, strongly suggesting how 3D structures arise at the tissue level. The clues proposed have opened the way to reproduce biomimetic, hierarchically ordered, assemblies of biological macromolecules. The present paper will focus on collagen and describe the process allowing to attain homogeneous fibrillar matrices, both at high concentrations and over long distances. Their characterization at scales ranging from μm to cm will demonstrate suprafibrillar arrangements similar to dense connective tissues, with aligned or helicoidal geometries depending on the protein concentrations. In addition we will show that in vitro and in vivo studies validate these tissue-like constructs as repair materials for tissue engineering applications.

Estrogen Receptor Expression in Posterior Tibial Tendon Dysfunction: A Pilot Study

The pathophysiology of posterior tibial tendon dysfunction (PTTD) is poorly understood. It has been theorized that changes in hormone physiology may be a factor influencing tendon health. Estrogen's influence on the fibroblast has been studied in other musculoskeletal tissues. Gender differences in anterior cruciate ligament (ACL) injuries have been studied and it has been discovered that the Estrogen receptor (ER) as well as Progesterone receptor (PR) are expressed in the ACL. Eight patients with PTTD requiring surgery were enrolled in our pilot study. The mean patient age was 52.4 (range, 18 to 73) years. There were five female and three male patients. Tendon samples were harvested from diseased PTT. Tendon samples harvested from healthy PTT and healthy flexor digitorum longus (FDL) tendon were used as controls. Tendon samples were processed using specific protocols for total RNA isolation from hypocellular, dense connective tissues. ERα and ERβ transcripts were quantified using real time RT-PCR. Quantitative values were obtained from the threshold cycle (Ct) number at which the increase in fluorescent signal associated with an exponential increase of PCR products can be detected. Transcripts of both ERα and ERβ were reproducibly detected in RNA samples isolated from our tendon samples. There was no difference in receptor expression between diseased and control tendon samples. There was no difference in receptor expression between male and female patients. We found that the tenocyte of the PTT and FDL tendons express ERα and ERβ. Normal and diseased tendons of both male and female patients expressed both estrogen receptors. Identifying ERα and ERβ gene expression in the fibroblast was an initial step in discovering whether tenocytes are targets for estrogen function. Estrogen receptors were identified indirectly by measuring receptor gene expression but we were unable to show a significant difference between diseased and control tendons.

Characterization of dense artificial connective tissues generated in a newly designed bioreactor

Dense connective tissues were generated simultaneously by accumulating collagen fibrils and fibroblasts on stainless steel mesh using a bioreactor system that we designed. The advantage of our system is that the artificial connective tissues can be generated within 24 hr in the absence of inhibitors against matrix metalloproteinases. The fibroblasts were suspended in 200 mL of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 0.5 mg/mL type I collagen. The mixed solution was circulated in two types of bioreactors with cylindrical or vertical configurations to generate luminal or parenchymal tissues, respectively. The gelatin zymography showed that MMPs were first detected in the media after 8 hr from the start of circulation and reached the highest levels on day 3. Glossy white aggregates, 1–3 mm in thickness, depending on the circulation period, accumulated on mesh grids. Fibroblasts were embedded in the network of collagen fibrils and possessed oval nuclei with or without prominent cell processes to form a bipolar shape. We could not observe distended cisternae of the endoplasmic reticula, the Golgi apparatus, or exploded mitochondria, showing hypoxic degenerative alterations of fibroblasts in dense connective tissues. The artificial tissues generated by our system will be useful for biological studies and transplantation therapy.

Functional tissue engineering of ligament healing

Ligaments and tendons are dense connective tissues that are important in transmitting forces and facilitate joint articulation in the musculoskeletal system. Their injury frequency is high especially for those that are functional important, like the anterior cruciate ligament (ACL) and medial collateral ligament (MCL) of the knee as well as the glenohumeral ligaments and the rotator cuff tendons of the shoulder. Because the healing responses are different in these ligaments and tendons after injury, the consequences and treatments are tissue- and site-specific. In this review, we will elaborate on the injuries of the knee ligaments as well as using functional tissue engineering (FTE) approaches to improve their healing. Specifically, the ACL of knee has limited capability to heal, and results of non-surgical management of its midsubstance rupture have been poor. Consequently, surgical reconstruction of the ACL is regularly performed to gain knee stability. However, the long-term results are not satisfactory besides the numerous complications accompanied with the surgeries. With the rapid development of FTE, there is a renewed interest in revisiting ACL healing. Approaches such as using growth factors, stem cells and scaffolds have been widely investigated. In this article, the biology of normal and healing ligaments is first reviewed, followed by a discussion on the issues related to the treatment of ACL injuries. Afterwards, current promising FTE methods are presented for the treatment of ligament injuries, including the use of growth factors, gene delivery, and cell therapy with a particular emphasis on the use of ECM bioscaffolds. The challenging areas are listed in the future direction that suggests where collection of energy could be placed in order to restore the injured ligaments and tendons structurally and functionally.