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Do cells in healing ligaments or tendons have deformation processes? If so, what are they?

Do cells in healing ligaments or tendons have deformation processes? If so, what are they?


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I am trying to better understand how non-muscle tissue in the muscuoloskeletal system heals. Specifically, I am interested in how ligaments or tendons heal once torn.

I read the following text:

If the muscle pulls the damaged tendon and then releases it then we have a deformation cycle that the cells are able to detect. They also then receive the information they require regarding orientation. This is why the elasticity of the early callus is important; the tissue must be able to be pulled out and then return to its original shape in order to receive information about orientation.

Source: http://www.peraspenberg.com/texts/how-do-tendons-and-ligaments-heal/

I don't understand what they mean by "deformation cycle" and how cells detect it.

Is there a name for this cycle? If so, how do the cells detect tissue deformation?

Also, if there is any literature that specializes in the tissue of ligaments or tendons, recommendations would be appreciated. I have plenty on muscle tissue and some on bones, but not ligament or tendon tissue regeneration.


Abstract

Knee ligament injuries are common, particularly in sports and sports related activities. Rupture of these ligaments upsets the balance between knee mobility and stability, resulting in abnormal knee kinematics and damage to other tissues in and around the joint that lead to morbidity and pain. During the past three decades, significant advances have been made in characterizing the biomechanical and biochemical properties of knee ligaments as an individual component as well as their contribution to joint function. Further, significant knowledge on the healing process and replacement of ligaments after rupture have helped to evaluate the effectiveness of various treatment procedures.

This review paper provides an overview of the current biological and biomechanical knowledge on normal knee ligaments, as well as ligament healing and reconstruction following injury. Further, it deals with new and exciting functional tissue engineering approaches (ex. growth factors, gene transfer and gene therapy, cell therapy, mechanical factors, and the use of scaffolding materials) aimed at improving the healing of ligaments as well as the interface between a replacement graft and bone. In addition, it explores the anatomical, biological and functional perspectives of current reconstruction procedures. Through the utilization of robotics technology and computational modeling, there is a better understanding of the kinematics of the knee and the in situ forces in knee ligaments and replacement grafts.

The research summarized here is multidisciplinary and cutting edge that will ultimately help improve the treatment of ligament injuries. The material presented should serve as an inspiration to future investigators.


Frequently Asked Questions About Stem Cells

1. What are stem cells and why do we hear so much about them?
Stem cells help to create new cells in existing healthy tissues, and may help repair tissues in areas that are injured or damaged. They are the basis for the specific cell types that make up each organ in the body. Stem cells are distinguished from other cells by a few important characteristics: they have the ability to self-renew they have the ability to divide for a long period of time and, under certain conditions, they can be induced to differentiate into specialized cells with distinct functions (phenotypes) including, but not limited to, cardiac cells, liver cells, fat cells, bone cells, cartilage cells, nerve cells, and connective tissue cells. The ability of cells to differentiate into a variety of other cells is termed multipotency. What scientists learn about controlling stem cell differentiation can become the basis for new treatments of many serious diseases and injuries.

2. What is the difference between embryonic and adult stem cells?
Some organs contain stem cells, called adult stem cells, that persist throughout life and contribute to the maintenance and repair of those organs. Not every organ has been shown to contain these cells, and generally adult stem cells have restricted developmental potential, in that their capacity for proliferation is limited and they can give rise only to a few cell types. Embryonic stem cells, by contrast, can divide almost indefinitely and can give rise to every cell type in the body, suggesting that they may be the most versatile source of cells for research and transplantation therapy.

3. Where do stem cells come from?
There are several sources of stem cells used in research. Embryonic stem cells are obtained from the inner cell mass of a blastocyst. The blastocyst is formed when the fertilized egg, or zygote, divides and forms two cells, then again to form four, and so on until it becomes a hollow ball of about 150 cells. The ball of cells, now called the blastocyst, actually contains two types of cells -- the trophoblast, and the inner cell mass. The inner cell mass contains the pluripotent stem cells that can be isolated and cultured. Stem cells are also found in differentiated tissues and organs throughout the body.

Often referred to as adult stem cells, or tissue-specific cells, they have not been identified in all tissues and organs, but in many cases they do exist and have a confirmed roll in repairing and maintaining tissue that has been injured or damaged by disease. The adult stem cells can be isolated from samples of the tissue, with the cells suspended in liquid and separated based on cell surface markers using fluorescence activated cell sorting (FACS). Blood from the umbilical cord of a newborn baby also contains blood stem cells and is often harvested and banked for future use, either for the benefit of research or for future treatments that the donor may require. The amniotic fluid is another rich source of stem cells that are multipotent and often more robust than stem cells derived by other means. Lastly, induced pluripotent stem cells (iPS cells) can be derived from the large pool of differentiated cells in the body (e.g. skin, fat, muscle, etc), which are transformed into an embryonic-like stem cell state.

4. What are induced pluripotent stem (iPS) cells?
Induced pluripotent cells are derived from somatic (adult, non-germline) cells, which have been reverted to an embryonic stem cell-like state. Like embryonic stem cells, iPS cells can be differentiated into any cell in the body, and are therefore considered pluripotent. The process of creating these cells, often referred to as "reprogramming," involves introducing a combination of three to four genes for transcription factors delivered by retroviruses into the somatic cell.

More recent methods have replaced and reduced the number of genes required for the transformation, used alternative delivery methods to get the genes into the cell, or sought to replace the genes with chemical factors. Cells can be taken from patients with specific diseases such as ALS, Parkinson's, or cardiovascular disease and induced to form iPS cells. Multiple uses can be derived from iPS cells when they are differentiated to more specialized cell types, including the development of assays for studying disease processes, scanning drug candidates for safety and effectiveness, or application to regenerative medicine.

5. How are adult stem cells obtained?
Adult stem cells are most commonly obtained from the outside part of the pelvis, the iliac crest. A needle is inserted in the iliac bone and bone marrow is withdrawn or aspirated through the needle. Several samples may be obtained from one area in this manner. The stem cells may then be separated from other cells in the marrow and grown or expanded in the laboratory. This may take from 7 to 21 days. When stem cells are placed in a specific tissue environment, such as bone, they become activated. As they divide, they create new stem cells and second generation, progenitor cells. It is the progenitor cells which may differentiate into newer cells with the same phenotype as the host tissue

6. Why do scientists want to use stem cells?
Stem cell researchers are hopeful that, in the future, a wide range of diseases and traumatic injuries will be cured by some application of cell therapy using stem cells. Currently, donated organs and tissues are used to replace lost or damaged tissue in many disorders. The great regenerative potential of stem cells has created intense research involving experiments aimed at replacing tissues to treat Parkinson's and Alzheimer's diseases, osteoarthritis, rheumatoid arthritis, spinal cord injury, stroke, burns, heart disease, and diabetes. While some success has been achieved with laboratory animals, a very limited number of experiments have been conducted on humans. These few experiments, however, have shown the great potential for stem cells. Scientists believe that a deep understanding of the complex phenomenon of stem cell differentiation will lead to a potential cure for serious medical conditions that are caused by abnormal cell division and differentiation, such as cancer and several growth and development disorders.

Another reason why stem cell biologists are excited about this field is that human stem cells could also be used to test new drugs. For example, new medications could be tested for safety by applying them to specialized cells differentiated from a stem cell clone. Cancer treatment, for instance, could benefit tremendously if anti-tumor drugs could be tailored to target the tumor stem cell.

7. What are some examples of musculoskeletal treatments using stem cells?
At this point, most musculoskeletal treatments using stem cells are performed at research centers as part of controlled clinical trials. Stem cell procedures are being developed to treat bone fractures and nonunions, regenerate articular cartilage in arthritic joints, and heal ligaments or tendons. These are detailed below.

Bone fractures and nonunions: In bone, progenitor cells may give rise to osteoblasts, which become mature bone cells, or osteocytes. Osteocytes are the living cells in mature bone tissue. Stem cells may stimulate bone growth and promote healing of injured bone. Traditionally, bone defects have been treated with solid bone graft material placed at the site of the fracture or nonunion. Stem cells and progenitor cells are now placed along with the bone graft to stimulate and speed the healing.

Articular cartilage: The lining of joints is called the articular cartilage. Damage to the articular cartilage can frequently lead to degeneration of the joint and painful arthritis. Current techniques to treat articular cartilage damage use grafting and transplantation of cartilage to fill the defects. It is hoped that stem cells will create growth of primary hyaline cartilage to restore the normal joint surface.

Ligaments and tendons: Mesenchymal stem cells may also develop into cells that are specific for connective tissue. This would allow faster healing of ligament and tendon injuries, such as quadriceps or Achilles tendon ruptures. In this instance, stem cells would be included as part of a primary repair process.

8. Why are doctors and scientists so excited about human embryonic stem cells?
Stem cells have potential in many different areas of health and medical research. To start with, studying stem cells will help us to understand how they transform into the dazzling array of specialized cells that make us what we are. Some of the most serious medical conditions, such as cancer and birth defects, are due to problems that occur somewhere in this process. A better understanding of normal cell development will allow us to understand and perhaps correct the errors that cause these medical conditions. Another potential application of stem cells is making cells and tissues for medical therapies.

Today, donated organs and tissues are often used to replace those that are diseased or destroyed. Unfortunately, the number of people needing a transplant far exceeds the number of organs available for transplantation. Pluripotent stem cells offer the possibility of a renewable source of replacement cells and tissues to treat a myriad of diseases, conditions, and disabilities including Parkinson's disease, amyotrophic lateral sclerosis, spinal cord injury, burns, heart disease, diabetes, and arthritis.

9. How hard is it for scientists make the cells into treatments?
It's hard work. First, cells must be coaxed into becoming the desired cell types. That process is called differentiation. For example, researchers have successfully used chemicals to turn embryonic stem cells into neurons, beating heart cells, insulin-producing islet cells and others. But the process of differentiation for the myriad cells in the human body is an extremely complicated one that scientists are only beginning to understand. Getting the cells to do what doctors want once they're inside the body is a huge challenge. Second, scientists have to find a way to prevent cells from being rejected by a patient's immune system. For some therapies, matching the cells to patients could be similar to the way doctors match bone marrow when performing transplants.

10. What are the obstacles that must be overcome before the potential uses of stem cells in cell therapy will be realized?
Some of the promise of stem cell therapy has been realized. A prime example is bone marrow transplantation. Even here, however, many problems remain to be solved. Challenges facing stem cell therapy include the following: Adult stem cells Tissue-specific stem cells in adult individuals tend to be rare. Furthermore, while they can regenerate themselves in an animal or person they are generally very difficult to grow and to expand in the laboratory. Because of this, it is difficult to obtain sufficient numbers of many adult stem cell types for study and clinical use. Hematopoietic or blood-forming stem cells in the bone marrow, for example, only make up one in a hundred thousand cells of the bone marrow. They can be isolated, but can only be expanded a very limited amount in the laboratory. Fortunately, large numbers of whole bone marrow cells can be isolated and administered for the treatment for a variety of diseases of the blood. Skin stem cells can be expanded however, and are used to treat burns.

For other types of stem cells, such as mesenchymal stem cells, some success has been achieved in expanding the cells in vitro, but application in animals has been difficult. One major problem is the mode of administration. Bone marrow cells can be infused in the blood stream, and will find their way to the bone marrow. For other stem cells, such as muscle stem cells, mesenchymal stem cells and neural stem cells, the route of administration in humans is more problematic. It is believed, however, that once healthy stem cells find their niche, they will start repairing the tissue. In another approach, attempts are made to differentiate stem cells into functional tissue, which is then transplanted.

A final problem is rejection. If stem cells from the patients are used, rejection by the immune system is not a problem. However, with donor stem cells, the immune system of the recipient will reject the cells, unless the immune system is suppressed by drugs. In the case of bone marrow transplantation, another problem arises. The bone marrow contains immune cells from the donor. These will attack the tissues of the recipient, causing the sometimes deadly graft-versus-host disease. Pluripotent stem cells All embryonic stem cell lines are derived from very early stage embryos, and will therefore be genetically different from any patient. Hence, immune rejection will be major issue. For this reason, iPS cells, which are generated from the cells of the patient through a process of reprogramming, are a major breakthrough, since these will not be rejected. A problem however is that many iPS cell lines are generated by insertion of genes using viruses, carrying the risk of transformation into cancer cells. Furthermore, undifferentiated embryonic stem cells or iPS cells form tumors when transplanted into mice. Therefore, cells derived from embryonic stem cells or iPS cells have to be devoid of the original stem cells to avoid tumor formation. This is a major safety concern. A second major challenge is differentiation of pluripotent cells into cells or tissues that are functional in an adult patient and that meet the standards that are required for 'transplantation grade' tissues and cells. A major advantage of pluripotent cells is that they can be grown and expanded indefinitely in the laboratory. Therefore, in contrast to adult stem cells, cell number will be less of a limiting factor. Another advantage is that given their very broad potential, several cell types that are present in an organ might be generated. Sophisticated tissue engineering approaches are therefore being developed to reconstruct organs in the lab. While results from animal models are promising, the research on stem cells and their applications to treat various human diseases is still at a preliminary stage. As with any medical treatment, a rigorous research and testing process must be followed to ensure long-term efficacy and safety.

11. When will stem cell research lead to new disease cures?
Adult stem cell-based therapies are already in widespread clinical use and have been for over 40 years, in the form of bone marrow transplants. These procedures, used to treat leukemia, lymphoma and inherited blood disorders, save many lives every year, and demonstrate the validity of stem cell transplantation as a therapeutic concept. New clinical applications are being explored using stem cells for the treatment of multiple sclerosis, cardiavascular disease, stroke, autoimmune and metabolic disorders, and chronic inflammatory diseases in addition to blood cancers. While human clinical trials have begun in many of these applications, it may still be a matter of years before these treatments become widely available to the patient. Nevertheless, we are optimistic that successes will be possible, and that new stem cell based treatments will become available as they complete clinical trials.

12. Are stem cell treatments safe?
That remains to be seen. Potential dangers include:

  • As stem cells renew themselves and can become different kinds of cells, they might become cancer cells and form tumors.
  • Stem cells grown in the laboratory, or adult cells reprogrammed to be stem cells, might have genetic damage.

There is also risk in some of the procedures used to get stem cells out of the body (such as from liposuction or spinal tap) or to deliver stem cells to the body (such as implanting them in the heart, brain, spinal cord, or other organs). That's not so much about the stem cells, but because of the procedures themselves. Researchers are studying all of that. Without carefully controlled clinical trials, there's no way to know what might happen in the long term, or even in the short term. That's why the FDA discourages the use of stem cells except in clinical trials or approved therapies. Every medical procedure has risks. A goal of clinical trials is to determine whether the potential benefit of a treatment outweighs the risks. A possible risk of some stem cell treatments may be the development of tumors or cancers. For example, when cells are grown in culture (a process called expansion), the cells may lose the normal mechanisms that control growth. A particular danger of pluripotent cells is that, if undifferentiated, they may form tumors called teratomas. Other possible risks include infection, tissue rejection, and complications arising from the medical procedure itself.

13. Are treatments using my own (autologous) stem cells safe? Why should these be regulated?
While your own cells are less likely to be rejected by your immune system, this does not necessarily mean the cells are safe to use as a therapeutic treatment. The methods used to isolate, modify, grow or transplant the cells may alter the cells, could cause infection or introduce other unknown risks. Transplanting cells into a different part of the body than they originated from may have unforeseen risk, complications or unpredictable outcomes.

14. What can I lose in trying an unproven treatment?
Some of the conditions that clinics claim are treatable with stem cells are considered incurable by other means. It is easy to understand why people might feel they have nothing to lose from trying something even if it is unproven. However, there are very real risks of developing complications, both immediate and long-term, while the chance of experiencing a benefit is likely very low. In one publicized case, a young boy developed brain tumors as a result of a stem cell treatment. Receiving an unproven treatment may make a person ineligible to participate in upcoming clinical trials. Where cost is high, there may be long-term financial implications for patients, their families and communities. If travel is involved there are additional considerations, not the least of which is being away from family and friends.

15. I have heard that there are clinics offering different types of stem cell treatments. Is this true?
Many clinics from all over the world offer stem cell therapies for a variety of diseases. However, many of these treatments are unproven, and in addition, these treatments tend to be very expensive.

16. Are there other uses of stem cells besides using them to treat disease?
Yes. Stem cells can be used to generate cell lines specific to a particular patient with a particular disease. By matching the biological data from these cells with the clinical history of the patient, it may be possible to extract more relevant information on the linkage between molecular pathways and the causes of disease. Cell lines can be derived from stem cells for specific tissues, such a heart muscle, specific types of neurons, kidney cells, etc. and used in biological assays to screen thousands of chemical compounds for their safety and effectiveness in treating disease. Stem cells also play an important role in expanding our understanding of embryonic and fetal development, helping us to identify the cells and molecules responsible for guiding the patterns of normal (and abnormal) tissue and organ formation.


Clinical classification of muscle strains

The clinical picture of a muscle strain depends on the extent and nature of the muscle destruction and the hematoma that develops at the site of the injury. In exercise induced strains the hematoma is most often intramuscular. The extravasated blood within the intact muscle fascia increases intramuscular pressure, which subsequently compresses the bleeding blood vessels and thereby eventually limits the size of the hematoma. In a severe strain the epimysium of the injured muscle may also rupture and then an intermuscular hematoma develops. DOMS may be considered the mildest form of strain injury, but since muscle fibers are not torn in DOMS, many do not regard it as a strain injury. Even the name is actually just a symptom, but since it is well known and widely used, we also employ it as a surrogate for the lacking pathogenetic term. Based on the clinical impairment muscle injuries have traditionally been classified as mild, moderate or severe6. Mild (grade I) strain represents a tear of only few muscle fibers with minor swelling and discomfort accompanied with no or only minimal loss of strength and restriction of the movements (ability to mobilize). Moderate (grade II) strain, in turn, is a greater damage of the muscle with a clear loss of function (ability to contract). Severe (grade III) strain occurs when a tear extends across the entire cross-section of the muscle (a very rare consequence of excessive intrinsic force alone) and thus results in a virtually complete loss of muscle function.

The traditional classification system described above does not take into account the exact location of the injury, which with the current capabilities and availability of modern imaging techniques such as magnetic resonance (MRI) and ultrasound (US) imaging, can now be exactly identified. Chan et al. 7 have recently described a new, very practicable classification system based on the findings on MRI or US investigation where the injury is defined as proximal, middle or distal by its location and then further defined as intramuscular, myofascial, myofascial/perifascial or myotendinous ( Tab. 1 ). Although the recent classification system by Chan et al. has to be applauded for its simplicity and practicability, injured skeletal muscle poses extra challenges for physicians beyond proper diagnostics and classification of the injury. Namely, it has been recently shown that similar looking skeletal muscle injuries in MRI at the same anatomical site, but caused by different mechanism of action (either high-speed running or over-stretching), have completely different healing rates 8, 9.

Table 1.

New clinical classification system for skeletal muscle injuries.

Site of lesion
1. Proximal MTJ
2. MuscleA. Proximala. Intramuscular
B. Middleb. Myofascial
C. Distalc. Myofascial/perifascial
d. Myotendinous
e. Combined
3. Distal MTJ

MTJ – Myotendinous junction


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Chen, L., Kim, P. D., Ahmad, C. S. & Levine, W. N. Medial collateral ligament injuries of the knee: current treatment concepts. Curr. Rev. Musculoskelet. Med. 1, 108–113 (2008).


NSAID EFFECTS ON TENDON-TO-BONE HEALING

The enthesis is the specialized junction between a tendon or ligament and bone (34). The enthesis progressively changes from tendon, to fibrocartilage, to calcified fibrocartilage, and finally bone (7). However, these four zones are not recreated following surgical repair but rather the tendon is joined to the bone through alternating layers of fibrovascular scar tissue (18, 41, 49, 78). This process requires chemotactic factors to guide inflammatory cells to the wound to initiate angiogenesis and scar formation, mitogenic factors to increase cell proliferation and scar matrix deposition by fibroblasts, and remodeling of collagen types I and III within the scar tissue to increase mechanical strength (41, 49). How NSAIDs affect each of these processes individually within the context of enthesis repair is not yet known. However, animal studies have shown that overall, NSAIDs appear to inhibit proper enthesis repair. Cohen et al. (22) showed that celecoxib and indomethacin treatment of an acute supraspinatus repair in rats resulted in inconsistent regrowth of a fibrocartilage zone between the tendon and the bone, whereas control specimens showed fibrocartilage formation by 4 wk and improved collagen fiber organization by 8 wk. Parecoxib and indomethacin treatment were shown to significantly lower the maximum pull-out strength and stiffness of Achilles tendons in rats that were reattached through a bone tunnel in the distal tibia (32). Additionally, celecoxib or indomethacin therapy reduced failure loads for rotator cuff repairs in rats (22). Histological analysis showed that there were substantial differences in collagen organization and maturation, which may have contributed to the decreased failure load of the treated animal. Similar conclusions were made when several different NSAIDs produced detrimental effects on healing strength at the bone-tendon junction of rat patellar tendon (37).

NSAID therapy likely affects inflammation after surgical repair of the enthesis, but NSAID therapy could also be directly affecting scar formation and remodeling. For instance, NSAIDs can impair osteoclast activity, and inhibition of osteoclast activity can enhance healing of rabbit anterior cruciate ligament repairs (79). Of note, Rodeo and colleagues (78) demonstrated that the strength of the interface between tendon and bone increases the most during the first 4 wk after surgery, which should be a consideration when using NSAIDs during the early stages of postsurgical recovery. These findings are not entirely surprising: since tendon-to-bone healing requires bone growth, it is possible that NSAIDs affect tendon-to-bone healing through a similar mechanism as fracture healing, since both processes require extensive bone metabolism.


The functions of entheses

Anchorage and stress dissipation

The attachment of a tendon/ligament to the skeleton is clearly the basic function of any enthesis and central to force transmission. Thus, tendons and ligaments often flare out at their attachment sites as an adaptation to securing skeletal anchorage and resisting the effects of insertional angle change. Flaring of entheses is particularly striking in the limbs, e.g. at the Achilles tendon, the insertion of peroneus brevis and the tibial attachment of the ACL. It is also an obvious but understated fact that most tendons and ligaments do not attach to the skeleton in an isolated manner. The enthesis of one often blends with that of another, so that many bony attachment sites overlap and this adds to the stability of the anchorage (Benjamin et al. 2004a). There are also many unnamed fibrous connections linking tendons and ligaments directly together near their attachment sites. Both these statements are in line with the concept of myofascial continuity explained in detail by Myers (2001). He proposes that what may appear to be discrete muscles are mechanically linked to each other by fascia that establish important lines of force transmission.

There are repeated misconceptions in the literature about how this anchorage is achieved. It is erroneous to think that this always hinges on ‘Sharpey's fibres’, yet the concept is deeply entrenched in the literature. Yes, some tendons/ligaments certainly do have Sharpey's fibres – notably fibrous entheses in regions where there is substantial cortical bone, at the sites where the periodontal ligament is attached to cementum and alveolar bone (Raspanti et al. 2000) and at the sites of surgical reattachment of tendons and ligaments to the skeleton (Johnson, 2005). Yet, a great many tendons and ligaments attach to areas of bone where there is virtually no cortex (short bones and the epiphyses and apophyses of long bones). At such sites, there seems little chance of deeply penetrating collagen fibres crossing the tissue boundary, although it does not completely negate the possibility that some fibres cross the divide. Such entheses are invariably fibrocartilaginous and collagen fibre continuity across the hard/soft tissue boundary occurs predominantly at the level of the tidemark which separates calcified and non-calcified fibrocartilage. It is perhaps these fibres which should be regarded as the functional equivalent of Sharpey's fibres in a fibrous enthesis. Milz et al. (2002) have suggested that it is the complex interdigitation of the layer of calcified fibrocartilage with the adjacent bone that secures attachment. Thus, there are parallels with the mechanism by which enamel and dentine are joined together in a tooth (Marshall et al. 2001). The dentinoenamel junction is markedly scalloped to increase the bonding between the tissues, and similar ‘scalloping’ occurs at fibrocartilaginous entheses. As there may be gradients of mineral content across the dentinoenamel junction that have a role in reducing stress concentration (Marshall et al. 2001), it would be interesting to know if the same applies to entheses.

There are intriguing parallels between entheses and numerous other biological interfaces, and these may add to our understanding of mechanical adaptations at insertion sites. As Waite et al. (2004) have eloquently stated, the widespread evolution of stiff scaffolds in the animal kingdom for 𠆏rame, integument, and appendages’ has created challenges in linking hard and soft tissues which nature has had to overcome with parallel adaptations. Thus, other biological interface regions presenting mechanical challenges which parallel those faced at entheses include the hard jaws of marine polychaete worms that anchor to soft tissue at their base, and the byssus threads which anchor mussels to rocky shores (Waite et al. 2004). It is worth noting that byssus threads are viewed as 𠆎xtracorporeal tendons’ (they are collagenous structures) and like long human tendons, they show regional differentiation along their length (Bell & Gosline, 1996).

It is also worth considering parallels between the mechanical anchorage of trees via their root systems and entheses. Like tendons and ligaments, plants are subject to mechanical forces created by their static loading, the influence of the wind, and the slope of the ground (Ennos et al. 1993). A remarkably small proportion of the plant actually participates in securing firm skeletal anchorage and it is clearly the same with tendons and ligaments. Thus, in both cases the necessary anchorage is created with a minimum investment in structural material. This maximizes the proportion of the tendon/ligament that can remain compliant and flexible and thus serve other functions (e.g. energy storage and changing the direction of muscle pull – Benjamin & Ralphs, 1998). Just as the lateral roots of a tree fan out from the point where the trunk meets the ground, equally the bony spicules beneath many entheses can radiate in all directions (Suzuki et al. 2005). There are other entheses (e.g. Achilles and patellar tendons) where there is considerable anisotropy of superficial trabeculae (Suzuki et al. 2005). Collectively, these may be likened to the tap root of a tree. Although the trabecular network is generally ignored when considering entheses, it obviously plays an important part in tendon/ligament anchorage and stress dissipation.

There are also useful comparisons to explore with non-biological composites. Humans are constantly seeking to join together materials of different physical properties (e.g. ceramic to metal) or to create surfaces that are resistant to contact damage (e.g. for magnetic storage media). What material scientists try to do is to create materials with graded mechanical properties that can then resist damage more effectively than their homogeneous counterparts (Suresh, 2001). This exactly parallels what fibrocartilage is believed to do at entheses. The presence of the two zones of fibrocartilage between the tendon/ligament ‘proper’ and the bone contributes to stress dissipation at entheses by ensuring that there is a gradual change in mechanical properties between hard and soft tissues (Woo et al. 1988). As Hems & Tillmann (2000) have emphasized, tendon and bone have similar tensile strength, but the elastic modulus of bone is approximately 10 times larger than that of tendon. Hence, a primary function of entheses must be to balance such widely different elastic moduli.

According to Suresh (2001), gradients at interface regions smooth stress distribution, eliminate singularities in stress, reduce stress concentration, improve the strength of the bonding and decrease the risk of fracture (i.e. failure). He also points out that the major mechanical difficulty which arises from joining a stiff scaffold (bone in the context of entheses) to a softer material (tendon or ligament) is 𠆌ontact deformation and damage’. Here, it may be useful to note Waite et al.'s (2004) metaphor of a wicker basket filled with blackberries. The fruit in contact with the basket walls is always the first to be damaged. It is the requirement for smooth stress distribution which accounts for why discrete (well circumscribed) entheses, at which the tendon/ligament attaches to a small, precisely localized region of bone, are fibrocartilaginous, and why this tissue is not a typical feature of entheses where the surface area of the junctional region is large (Thomopoulos et al. 2003).

The paucity of attempts to study biomechanical aspects of entheses in relation to junctional properties is largely because of practical difficulties of recording strain levels within such a small volume of tissue and the transitional nature of the region with no clear boundaries that define it. Maganaris et al. (2004) argue that loading on entheses is non-uniform across the attachment site and they cite several studies which all show that the pathology occurs in the regions where strain levels are lowest. They make the interesting suggestion that the regions most vulnerable to damage at entheses are initially stress-shielded and that tensile failure may not be a key feature of enthesopathy. They have rightly drawn attention to the fact that clinically recognizable enthesopathy occurs more frequently in the deep than the superficial part of an enthesis. This corresponds with a regional difference in the prominence of enthesis fibrocartilage – which is generally more conspicuous in the deepest parts of entheses (Benjamin et al. 1986 Woo et al. 1988). Fibrocartilage is an adaptation to compression and/or shear (Benjamin & Ralphs, 2004) and the deep part of an attachment site is compressed by the superficial part. It is these compressive forces that may be pertinent to understanding enthesopathies.

In the early 1990s, Benjamin and colleagues published a series of papers which all suggested that there was a correlation between the quantity of uncalcified fibrocartilage at an enthesis and the degree of ‘insertional angle change’ that occurred during joint movement (Evans et al. 1990 Benjamin et al. 1991, 1992). An ‘insertional angle change’ is the change in the angle at which the tendon/ligament meets the bone as the joint(s) is moved. It is suggested that the stiffened ECM that typifies the fibrocartilaginous region of a tendon/ligament promotes the gradual bending of the collagen fibres as they approach the hard tissue interface. This function of enthesis fibrocartilage has often been compared with that of a grommet on an electrical plug – an analogy first used by Schneider (1956).

Although tendons and ligaments are often viewed as non-distensible, they do have the ability to stretch and recoil by approximately 6% of their original length without any obvious signs of damage. It is in recognition of this that Knese & Biermann (1958) proposed their ‘stretching-brake theory’ of enthesis fibrocartilage function. These authors pointed out that the stiffened ECM at a fibrocartilaginous enthesis should limit the narrowing of an elongated tendon/ligament at this region. The theory is an attractive one, but largely ignored by subsequent authors and thus one which remains unsubstantiated. Nevertheless, Milz et al. (2005) have suggested that a stretching brake function could well operate at the entheses of the human acetabular ligament. This is a very short ligament, which has conspicuous fibrocartilaginous entheses, yet exhibits little insertional angle change with joint loading. They argue that the rapid increase of tensile stress on this ligament during load bearing is likely to produce a biologically relevant shear stress that acts as the mechanical stimulus for fibrocartilage formation.

Entheses as mini growth plates

Knese & Biermann (1958) have suggested that entheses can act as growth plates for apophyses at tendon and ligament attachment sites. This is supported by the developmental study of Gao et al. (1996) on the femoral attachment of the medial collateral ligament of the rat knee joint. Gao et al. (1996) exploited age-related changes of labelling in types I and II collagen to show that the cartilage at the enthesis is initially derived from that of the embryonic bone rudiment. Equally, however, they showed that this hyaline cartilage is eroded during endochondral ossification and replaced by enthesis fibrocartilage that develops within the adjacent ligament by fibroblast metaplasia. Despite this, little is known about the molecular control of cell maturation at entheses. Given the similarities with the growth plate, the terminal differentiation of enthesis fibrocartilage cells invites comparison with the control of chondrocyte differentiation in epiphysial growth plates. This is a multistep process regulated by a complex network of signalling systems. Early stages involve control of chondrocyte proliferation, followed by hypertrophy and apoptosis, angiogenesis and osteogenesis. Proliferation, which in the growth plate is regulated by IGF (e.g. Olney & Mougey, 1999), appears not to be a major issue at the enthesis, as there is no evidence for enhanced tendon or fibrocartilage cell proliferation (Woo et al. 1988). However, control of the later stages is significant. In the epiphysial growth plate, this involves a complex interaction of a variety of signalling molecules, some of which are also involved in early tendon, cartilage and fibrocartilage differentiation as indicated above. Cessation of proliferation and onset of hypertrophy is stimulated by FGFs and BMPs (Volk et al. 1998 de Crombrugghe et al. 2000), with a negative feedback regulation of hypertrophy and enhancement of proliferation provided by Ihh produced by hypertrophic chondrocytes acting on the perichondrium to produce PTHrP (de Crombrugghe et al. 2001 Vortkamp, 2001). At the transcriptional level, hypertrophy is regulated by Cbfa1 (Leboy et al. 2001 Takeda et al. 2001). Although there are differences in the organization of tissues and cells, the similarities are sufficient to make it important to discover the extent to which controls operating in the growth plate also occur in the enthesis.

Following fibrocartilage ‘hypertrophy’, angiogenesis and osteogenesis occur following the erosion of the terminal ‘hypertrophic’ fibrocartilage cells of the enthesis (Benjamin et al. 2000). Nothing is known about how this happens at entheses, but again the analogy is with growth plates. Chondrocyte hypertrophy is followed by apoptosis and vascular invasion, fibrocartilage cells express hypertrophic markers and then undergo regulated cell death (Yamada, 1976) before the space they occupy is invaded by blood vessels. In cartilage, hypertrophic chondrocytes express VEGF (Gerber et al. 1999 Colnot & Helms, 2001), the major stimulator of angiogenesis, and expression is modulated by cartilage-promoting growth factors FGFa FGFb, TGF beta and IGF-1 (Garcia-Ramirez et al. 2000 Gerber & Ferrara, 2000). GDF5, which is associated with tendon differentiation and has been linked to fibrocartilage formation (Bostrom et al. 1995 Takae et al. 1999 Nakase et al. 2001), also has angiogenic effects (Yamashita et al. 1997). VEGF is bound to cartilage ECM, and can be released by matrix metalloproteinases (MMPs) (Vu et al. 1998 Gerber et al. 1999), so the expression of these is clearly important in growth plate angiogenesis. Mt1-MMP and MMP9 are expressed by the perichondrium in developing cartilage rudiments, whereas MMP13 occurs in late hypertrophic cells, and is important in the differentiation process – inhibition of collagenase arrests hypertrophy (Kim et al. 1999 Colnot & Helms, 2001 Wu et al. 2002). VEGF itself can also have direct effects on osteoblast differentiation (Deckers et al. 2000), thus linking angiogenesis to osteogenesis. Thus, it is clear that determining the control of angiogenesis in the enthesis is important in understanding its development and growth.


Stem Cells and Platelet-Rich Plasma Enhance the Healing Process of Tendinitis in Mice

Objective. Achilles tendon pathologies occur frequently and have a significant socioeconomic impact. Currently, there is no evidence on the best treatment for these pathologies. Cell therapy has been studied in several animal models, and encouraging results have been observed with respect to tissue regeneration. This study is aimed at evaluating the functional and histological effects of bone marrow stem cell or platelet-rich plasma implantation compared to eccentric training in the treatment of Achilles tendinopathy in rats. Methods. Fourty-one male Wistar rats received collagenase injections into their bilateral Achilles tendons (collagenase-induced tendinopathy model). The rats were randomly divided into four groups: stem cells (SC), platelet-rich plasma (PRP), stem cells+platelet-rich plasma (SC+PRP), and control (eccentric training (ET)). After 4 weeks, the Achilles tendons were excised and subjected to biomechanical and histological analyses (Sirius red and hematoxylin-eosin staining). Results. Biomechanical assessments revealed no differences among the groups in ultimate tensile strength or yield strength of the tendons (

), but there were significant differences in the elastic modulus (MPa

) and maximum tensile deformation ( ). The PRP group showed the greatest maximum deformation, and the SC group showed the highest Young’s modulus (elasticity) measurement. In histological analysis (hematoxylin-eosin and Sirius red staining), there were no differences among the groups. Conclusion. PRP and SC+PRP yielded better biomechanical results than eccentric training, showing that these treatments offer better tend function outcomes. This theoretical rationale for the belief that cell therapies can serve as viable alternatives to current treatments chronic fibrotic opens the door for opportunities to continue this research.

1. Introduction

Tendon disorders are common in clinical practice and can cause significant morbidity, pain, and consequent practical reductions in both work and physical activity [1]. Because tendons are poorly vascularized [2], their healing potential is poor if they are damaged by acute or chronic lesions [3]. Work overload can cause them to undergo fibrosis-related structural changes. This situation predisposes patients to chronic pain and tendon rupture [4].

Achilles tendon pathologies are especially common and tend to be symptomatic [4]. Their prevalence varies and depends on the type and intensity of specific sports activities. The prevalence of Achilles tendinopathies can reach 66% among runners [5].

The use of platelet-rich plasma (PRP) as a treatment for tendinitis promotes cell proliferation and chemotaxis. PRP also enhances the healing potential by stimulating neovascularization and activating growth factors that increase gene transcription and protein synthesis. These changes trigger cell proliferation and cell differentiation, resulting in faster and more effective healing [6].

Nonetheless, in orthopedics, the results have not been so encouraging. Studies on the isolated administration of growth factors have shown more favorable results than studies on PRP administration [6]. However, there are still no randomized controlled trials to prove this notion [7].

As for bone marrow mononuclear stem cells, their ability to differentiate into multiple conjunctival cell types, including chondrocytes [8, 9] and tenocytes [10, 11], has already been well characterized in preclinical studies. Moreover, it is well known that stem cells (SCs) can induce the formation of linear arrangements of type I collagen [12] which increases the elastic modulus, yield strength, and resistance to deformation and improves the biomechanical characteristics of the tendon [13, 14].

SCs have been used experimentally for the treatment of superficial flexor tendinitis in horse toes, which resulted in significantly lower recurrence rates than those in horse toes that were subjected to standard treatments [15–17]. Nevertheless, these results are not persistent, because 4 months after injury, the recurrence rates are similar between the two groups [15].

In the present experimental rat study, our objective was to verify the functional and histological effects of injecting SC, PRP, or SC+PRP compared with eccentric training (ET) protocols in treating Achilles tendinitis induced by type I collagenase.

2. Materials and Methods

This was an experimental study performed according to the guidelines of the Brazilian College of Animal Experimentation (COBEA) and approved by the Ethics Committee (Pontifical Catholic University of Paraná (CEUA PUCPR), under ID number 01037). To assess these effects of the SC, PRR, SC+PRP, and ET protocols, we performed a histological analysis using hematoxylin-eosin and Sirius red staining. Biomechanical evaluations were also performed 4 weeks after treatment on the 41 Wistar male rats weighing between 250 and 350 g used in the study. All animals were subjected to bilateral percutaneous applications of type IA collagenase in the Achilles tendon regions, by clinical palpation with the ankle flexed at 90°, at doses of 250 IU (30 μl) and concentrations of 10 mg/ml [16], dissolved in 0.09% saline solution for a volume of 0.5 ml, after the intraperitoneal (IP) administration of a combined anesthesia regimen of 5% ketamine (80 mg/kg) and 2% xylazine hydrochloride (10 mg/kg). Five days after the injuries, the animals were randomized into 4 groups: SC group (

) (the rats were reanesthetized to administer SCs or PRP in groups SC, PRP, and SC+PRP), and an eccentric exercise protocol group (ET, control group,

SCs were obtained by collecting

2 ml of blood from the rats’ iliac crests. The blood was then separated by Ficoll density gradient centrifugation using IMDI culture medium (Iscove’s modified Dulbecco’s medium) supplemented with antibiotics (1% penicillin and streptomycin), according to the technique described by Boyum [17]. The SC specimens were resuspended and applied alone percutaneously to the animals’ bilateral Achilles tendons.

PRP was prepared by collecting

1-2 ml of blood via cardiac puncture according to Anitua’s technique. This was followed by serial homogenization and centrifugation [18]. Similarly, the specimens obtained from each animal for the PRP preparations were reapplied alone percutaneously to the Achilles tendons after resuspension.

For animals in the SC+PRP group, the two steps described above were carried out concomitantly. The animals in the ET group started an exercise training protocol on individual 15° inclination treadmills (Figure 1) following an adaptation regimen as follows: 1 km/h for 15 minutes in the first 2 weeks, gradually increasing to 45 minutes, followed by 17 m/min (1 km/h) for 1 hour three times a week for 2 additional weeks [19].

The animals were administered a subcutaneous dose of carprofen analgesic at 5 mg/kg per day via abdominal application for 72 hours after the implant procedure. The animals were killed 4 weeks later to comparatively analyze the groups. One tendon was analyzed histologically, and the contralateral tendon was subjected to biomechanical testing.

2.1. Functional Analysis

The biomechanical evaluations were performed on an EMIC DL500 device, which was used to assess the parameters of ultimate tensile strength, yield strength, maximum deformation, and elastic modulus.

2.2. Histology

For the histological evaluation, the Achilles tendons were mounted as 5 μm thick sections on histological slides and stained with hematoxylin and eosin to characterize the cells and extracellular matrix. Cellularity, vessels, and collagen fibers were scored from 1 (normal) to 4 (significant changes). The criteria to evaluate the tendons included the following: tenocyte morphology and density presence of hemorrhage, neovascularization, and inflammatory infiltrates linearity and undulation of collagen fibers and epitendon thickness (according to previous publications on histological evaluations of tendinopathy, as described by Urdzikova et al.) [20].

To evaluate the proportions of type I and III collagens in the affected tissue, the slides were stained with Sirius red, and a computerized analysis was carried out to assess the percentages. This approach, as reported by Nixon et al., has been used in other studies to evaluate tendinopathy [21].

2.3. Statistical Analysis

Quantitative variables were compared using the analysis of variance (ANOVA) or the Kruskal–Wallis test, whereas normality was assessed using the Shapiro–Wilk test. For qualitative variables, a Fisher or Chi-square test was conducted. Any value < 0.05 indicated a statistical significance (data were analyzed using IBM SPSS Statistics software, v.20.0 IBM Corp., Armonk, NY, USA).

3. Results

Four weeks after treatment, we obtained the following specimens for the histological analyses: (i) 8 tendons each from the SC and PRP groups (ii) 6 tendons from the SC+PRP group (iii) 9 tendons from the ET (control) group

For the biomechanical analyses, we obtained the following specimens: (i) 10 tendons each from the SC and PRP groups (ii) 12 tendons from the SC+PRP group (iii) 9 tendons from the ET (control) group

3.1. Functional Analysis

Regarding biomechanical testing, descriptive statistics of each parameter are presented in Table 1.

An outlier was excluded (equal to -5.14). SC: stem cell PRP: platelet-rich plasma ET: eccentric training ANOVA: analysis of variance.

Regarding ultimate tensile strength and yield strength, there were no statistically significant differences among the groups ( ). However, differences were observed in the maximum deformation (Figure 2) and elastic modulus (Figure 3) ( and , respectively) when the groups were compared pairwise (post hoc least significant difference (LSD) test,

). Table 2 below shows the values for these comparisons.

. PRP: platelet-rich plasma SC: stem cell: sd: standard deviation se: standard error.

. MPa: elastic modulus SC: stem cell PRP: platelet-rich plasma sd: standard deviation se: standard error.

Post hoc LSD (least significant difference) test

. MPa: elastic modulus SC: stem cell PRP: platelet-rich plasma ET: eccentric training.

The PRP group had significantly better results for maximum deformation and elastic modulus than all the other groups. The SC group had an elastic modulus result that was significantly higher than that of the other groups (Figures 2 and 3).

3.2. Histological Analysis

The tendons were prepared by staining the tissue sections with hematoxylin and eosin. Next, they were evaluated on a scale of 1 to 4 (where

abnormal) according to the following parameters: morphology and density of tenocytes, presence of hemorrhage, neovascularization, inflammatory cell infiltrate, linearity and undulation of collagen fibers, and epithelial thickness. Using this scoring system, each tissue section was scored between 8 (normal) and 32 (maximum abnormality), and the results were used in the statistical analysis.

After analyzing the score results, there were no statistically significant differences in histological scores among the groups ( Kruskal–Wallis test). The scores varied from 10 to 21 in the SC group (

), from 13 to 20 in the PRP group ( ), from 14 to 19 in the SC+PRP group ( ), and from 11 to 21 in the control (ET) group (

. SC: stem cell PRP: platelet-rich plasma ET: eccentric training.

Bleeding was not observed in any of the tissue sections in any group. All tissue sections in all groups received a score of 2 (slight increase) for neovascularization, with the exception of one tissue section belonging to the SC group, which received a score of 1 (none) (Table 3).

value: 0.411 (Kruskal-Wallis test). SC: stem cell PRP: platelet-rich plasma ET: eccentric training.

The inflammatory cell infiltrates received score 1 (not present) or 2 (slight increase) among groups, as illustrated in Table 4.

Regarding the linearity of the collagen fibers, the scores also varied only in 1 (normal) or 2 (more than 50% of linear collagen fibers) among groups (Table 5).

Undulation of the collagen fibers varied from normal (score value 1: all fibers are undulated) to moderately abnormal (score value 3: without undulation) among the groups (Table 6).

The thickness of the epitendons (Figures 5(a)–5(c)) also showed no significant differences among the groups however, no group exhibited evidence of normal epitendon organization. Two tissue sections showed maximum abnormalities in the SC group, as presented in Table 7.

There were no significant differences in tenocyte morphologies among the groups ( ), as described in Table 8 and Figures 6(a)–6(c).

value: 0.595 (Chi-square test). SC: stem cell PRP: platelet-rich plasma ET: eccentric training.

There was a tendency toward histological normality of the tenocyte densities in groups SC, PRP, and SC+PRP compared with that in the control group (ET) (Kruskal–Wallis test, and , respectively). In groups SC, PRP, and SC+PRP, the tissue sections scored 2 or 3 for this parameter, which meant a mild or moderate increase in tenocyte density, respectively. In the control group, this parameter scored 4 (tenocyte layers) (Table 9).

value: 0.073 (Chi-square test), SC: stem cell PRP: platelet-rich plasma, ET: eccentric training.

To assess the percentages of type I and III collagens in each group, the tissue sections were stained with Sirius red and analyzed using a software program that calculated the percentage of each collagen in the tissue sections.

The mean percentage of type I collagen in the SC group was 61.82, and the median value was 64.24 in the PRP group, the mean was 41.18 and the median was 39.71 in the SC+PRP group, the mean was 49.29 and the median was 44.28 in the ET group, the mean was 40.09 and the median was 34.25, as shown in Table 10 and Figure 7(a).

Regarding type III collagen, the mean percentage in the SC group was 38.17 and the median value was 35.76 in the PRP group, the mean was 58.81 and the median was 60.28 in the S+PRP group, the mean was 50.70 and the median was 55.72 in the control group (ET), the mean was 59.90 and the median was 65.74, as demonstrated in Table 11 and Figure 7(b).

Nonparametric Kruskal–Wallis test. SC: stem cell PRP: platelet-rich plasma ET: eccentric training.

There were no significant differences in the percentages of type I and III collagens among the groups (Kruskal–Wallis test, ) (Figures 6(a)–6(c)).

4. Discussion

To date, several studies have compared various conservative modalities used to treat Achilles tendinitis. However, the results are conflicting, indicating that nonhormonal and hormonal anti-inflammatory treatments might have efficacies similar to placebo. Hormonal anti-inflammatories are associated with Achilles tendon rupture when applied to peritendinous regions [8].

Likewise, sclerosing agents such as polidocanol act to prevent neovascularization and alleviate pain, but they are also associated with higher rates of tendon rupture [8]. Unfortunately, surgical treatments are associated with high failure rates [8].

The aforementioned reasons coupled with socioeconomic factors, especially with respect to injuries in elite athletes, have made Achilles tendinitis the target of several animal studies conducted to identify newer, more effective, and safer therapies.

Our data revealed that cell therapy, especially in the groups that received PRP and the SC+PRP combination, was associated with maximum deformation in biomechanical testing. The difference was significantly greater in these two groups than that in the other groups. This parameter is extremely important in the evaluation of Achilles tendinitis because the deformation capacity of tendons (elastic property) forms the basis for proper physiological function. These findings are consistent with other research data, such as that of Nixon et al., whose study involved PRP and tenocytes derived from adipocytes that were used in an animal model of tendinitis (also see Shah et al.’s) [21, 22]. Both studies revealed that the use of growth factors derived from PRP improved the biomechanical characteristics of injured tendons. Our findings corroborate the notion that growth factors derived from PRP are capable of stimulating new tissue formation in these pathological conditions [2, 21, 22].

Our study did not show a statistically significant difference among the groups in the histological evaluations. In contrast to other existing studies, our study compared the groups treated with cell therapies to a group treated with a physiotherapeutic modality not to an injured group that received no treatment. This approach might explain the smaller histological differences observed among our groups. In addition, the absence of histological and biomechanical differences among the groups has been demonstrated in the literature. Shah et al. revealed better biomechanical performances even in the absence of histological differences [22].

This phenomenon was also explained by Zhang et al. They reported a reduction in the expression of the COX-1 and COX-2 genes and lower levels of prostaglandins (e.g., PGE2) when rats were treated with PRP in a tendinitis model. In that study, they proved the anti-inflammatory effect of PRP.

The absence of histological and immunohistochemical differences was also mentioned by Parafioriti et al. [23]. In their surgical model of Achilles lesions in rats, although there were significant histological differences 1 week after treatment, they did not persist at 2, 4, and 6 weeks after treatment [23].

In contrast, our data showed a trend toward significant changes in histological tenocyte density in the cell therapy groups. In similar studies with larger sample sizes, the best organization of the extracellular matrix in stem cells groups has already been proven [20].

In addition to obtaining similar results, Crovace et al. showed that SC implantation restored the proportion of type I and III collagens compared to that in controls, thereby proving that tissue regeneration occurs after collagenase-induced tendon injuries in sheep [12].

In accordance with the literature, our data suggest that cell therapies may be more effective than currently available treatments. We have shown a higher efficacy of PRP in the treatment of tendons based on biomechanical test results and tendency toward histological normality of the tenocytes in groups that received cell therapies (SCs and/or PRP). This finding strengthens the notion that factors promoting cell differentiation can reactivate relatively inert cells such as tenocytes and assist in managing pathologies with predominantly fibrotic characteristics. A statistical extrapolation of the data obtained in this study indicates that a larger sample size would likely achieve higher levels of significance.

Despite the animal models indicating a superiority of cell therapy models in the treatment of tendinitis, a recent systematic review reported that randomized clinical trials are necessary for validation. In humans, relevant studies are currently limited to case series, which do not provide strong evidence for direct clinical application and point the need for clinical studies. Nevertheless, cell therapies, which promote medical tourism, are already available in some countries [24, 25].

Our findings support the importance of follow-up research and the need to improve this experimental model. Altogether, these findings offer much hope for effective and lasting treatment of these common and disabling pathologies.

This study is limited by the fact that it is an animal model. Additionally, there is no current standard treatment for Achilles tendinopathy to be used for comparison with potentially new therapeutic modalities.

5. Conclusion

We can conclude that, in a rat model of tendinitis, the functional effect of implanted PRP was stronger than that of SC alone, ET, or the SC+PRP combination at 4 weeks after treatment.

Regarding the histological analysis with hematoxylin and eosin alone, there was no statistically significant difference among the groups, either in the total score (sum of the scores of each item analyzed) or in separate analyses of each of the variables including tenocyte morphology and density, presence of hemorrhage, inflammatory cell infiltration, neovascularization, linearity and undulation of collagen fibers, and epithelial thickness.

In the analysis of the percentages of type I and III collagens determined by Sirius red staining, there were no statistically significant differences among the groups at 4 weeks after treatment.

Finally, it was the functional analysis of the biomechanical parameters that revealed a higher efficacy of PRP cell therapy compared to the treatments in the other groups. Tendon samples in the PRP group withstood the greatest deformation when compared to those in the other groups.

Accordingly, there is a theoretical rationale for the belief that cell therapies can serve as viable alternatives to the current treatments for chronic fibrosis disorders. This opens the door for opportunities to continue this research.

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

This project was funded by the researcher’s private funds and institutional funds from the Pontificia Universidade Católica do Paraná (PUCPR), Rua Imaculada Conceição 1155, 80215-901 Curitiba, PR, Brazil. No external funding support was received.

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Copyright

Copyright © 2019 Rosangela Alquieri Fedato et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


CONCLUSIONS

Most commonly, tendon injuries are chronic, resulting from cumulative “microtrauma,” degeneration (tendinosis), and ultimately tendon failure. 49 Clinically, surgical intervention for management of tendon tears is indicated only after failure of non-surgical approaches. 50, 51 The response of the fatigue damaged tendon to exercise as a therapeutic measure after damage is largely unknown. This makes it difficult for the clinician to have any scientific basis for treatment recommendations. In addition, since tendinopathies ultimately progress to ruptures, the limited success of surgical repair and high rates of retear 52, 53 have motivated our studies of scarless tendon healing using the MRL/MpJ mouse. While most of our work has focused on the basic mechanisms of regeneration of large tendon defects (biopsy punch), ongoing studies are evaluating the regenerative capacity of MRL/MpJ in more clinically applicable rotator cuff injuries. 54 Furthermore, our ongoing studies that are isolating the contributions of the innate tendon and the systemic environment to scarless tendon healing will identify new therapeutic pathways to promote scarless healing. For instance, the potential utility of MRL/MpJ's provisional ECM as a therapeutic intervention that harnesses the biological and structural mechanisms that lead to scarless tendon healing could be highly impactful for the field of tendon tissue engineering.


Supporting information

S1 Table. Summary of the literature data used for designing the strain-dependent collagen production laws.

In vitro strain-stimulation of fibroblasts from various sources. Collagen type 1 and 3 production is expressed as a relative increase to collagen content levels measured without strain stimulation measured after 12–48 hours of strain stimulation.

S1 Fig. Variations tested for strain-dependent tissue production law 2.

Three different center transition points (12.5%, 15.0% and 17.5%) and steepness (ksig = 37.5, 75, 150) parameters were tested.

S2 Fig.

The spatio-temporal evolution of collagen content (% of intact) at week 1, 2 and 4 upon perturbations in the center transition point (low: 12.5%, default: 15.0% and high: 17.5%) and steepness (ksig—low: 37.5, default: 75, high: 150) in production law 2. The width of the stumps is denoted by the black dotted lines. All perturbations of the model parameters predict a decrease in tissue production in the tendon core at week 1.

S3 Fig. The temporal evolution of tendon stiffness at week 1, 2 and 4 upon perturbations in the center transition point and steepness of production law 2, compared to the experimental data from Khayyeri et al. (2020).

Overall, all different models predicted a development of stiffness in the range of the experimental data. Note that the daily loading simulation for the model with center transition point (12.5%) did not converge beyond 12 days of healing, yet the predicted stiffness was well within the range of experimental data.

S4 Fig. The temporal evolution of mean tissue alignment (see Eq 11) in the healing tendon callus for the default reorientation rate (κ = 0.06, see Eq 10) for the model with production law 2.

S5 Fig. Parameter sensitivity of the speed of reorientation (see Eq 10) for the model with production law 2.

Three reorientation speeds (κ = 0.03–0.06–0.09) were evaluated, such that the majority of reorientation is completed in 6, 4 and 2 weeks, respectively. For each reorientation rate, the evolution of spatio-temporal evolution of collagen content and temporal evolution of stiffness is shown, all with production law 2. The width of the stumps is denoted by the black dotted lines. All models predicted decreased collagen content in the tendon core at week 1 of healing. Additionally, this effect was more prominent and persistent for the slowly reorienting model (κ = 0.03). The stiffness increased with increasing reorientation speed, but the predicted stiffnesses remained within the range of experimental data (Khayyeri et al., 2020).

S6 Fig. The spatio-temporal evolution of collagen content (% of intact) at week 1, 2 and 4 upon perturbations in cross-sectional area (1X and 2X CSA) and stump overlap (0-50-100%) for the model with production law 2.

When increasing the stump overlap, the collagen production becomes more homogeneous.

S7 Fig. The spatio-temporal evolution of the average collagen orientation with respect to the longitudinal axis at week 1, 2 and 4 upon perturbations in cross-sectional area (CSA) and stump overlap for the model with production law 2.

The spatio-temporal patterns of reorientation were similar in all models.

S8 Fig. The spatio-temporal evolution of collagen content (% of callus content) across the midtendon cross section at week 1, 2 and 4 upon perturbations in cross-sectional area (CSA) and stump overlap for the model with production law 2.

The width of the stumps is denoted by the black dotted lines. Decreased content in the tendon core is denoted by a red arrow.

S9 Fig. The spatio-temporal evolution of collagen content (% of intact) across the midtendon cross section and for the whole callus at week 1, 2 and 4 for the default and 50% longer callus for the model with production law 2.

The width of the stumps is denoted by the black dotted lines. Decreased content in the tendon core is denoted by a red arrow. The model with increased callus height predicted a more homogeneous collagen production.

S10 Fig. The spatio-temporal evolution of the average collagen orientation at week 1, 2 and 4 for the default and 50% longer callus for the model with production law 2.

The model with increased callus height displayed a very similar evolution of the spatial distribution of collagen alignment.

S11 Fig. Long-term prediction of collagen content, collagen alignment, overall tendon stiffness and the relative change in these properties for the model with production law 2.

To characterize the long-term predictions of the current healing framework, the model with production law 2 ran for 100 days (

14 weeks). All monitored properties (mean tissue content, alignment, and stiffness) approached steady-state within 7 weeks of healing. The dashed line represents 5% relative change. Within 4 to 7 weeks, all properties changed less than 5% with respect to previous week.


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Comments:

  1. Ring

    It's the right information

  2. Dilrajas

    My God! Well well!

  3. Tanton

    brute force)



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