Page 1 of 11

Critical review

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F�� �������� ��������: Moshiri A, Oryan A. Role of tissue engineering in tendon reconstructive surgery and regenerative medicine: current concepts, approaches and concerns. Hard Tissue. 2012 Dec 29;1(2):11.

Role of tissue engineering in tendon reconstructive surgery and regenerative medicine: current concepts, approaches

and concerns

A Moshiri1*, A Oryan2

AbstractIntroductionSurgical reconstruction of tendon inj-uries is challenging. Classic reconstr-uctive techniques and tendon transp-lantation have some significant limit-ations and tissue engineering is a ne-wer option. Despite significant devel-opment in tissue engineering techno-logies, the role of tissue engineering in tendon healing is still unclear. Res-earchers have also focused on in vitr-o investigations and because of the differences between ex vivo and in v-ivo situations, translation of their re-sults to clinical practice is of great c-oncern and generally hard to follow. To increase the impact of tissue engi-neering in tendon healing, more info-rmation concerning the structure of tendons, their injuries, healing and host immune response together with the characteristics of biomaterials is needed to produce a more effective tissue-engineered product with the aim to substitute the classic reconst-ructive methods with the new tissue engineering approaches. This review was aimed to introduce the most im-portant issues in the relationship be-tween tissue engineering and tendon regenerative medicine with the hope that this information would be valua-

ConlusionTendon tissue engineering is still in its infancy and translation to clinics is accompanied by several important concerns including graft healing incorporation, host immune reaction and in vivo efficacy of the tissue-engineered graft.

IntroductionTreatment of tendon injuries is chall-enging1,2. Classic surgical reconstruc-tive methods have significant limitat-ions with unclear outcomes, especial-ly in tendon injuries having-large tendon deficits3,4. Tendon transplant-ation is the only available option wh-en the injured tendons cannot be re-paired with classic surgical techniqu-es2,3. Natural grafts can be divided in-to three major groups including auto-grafts, allografts and xenograft-s2,5,6. All of them have their own sign-ificant limitations. For autografts, th-ese limitations include availability of the autograft of the same size, shape and physiological characteristics of t-he normal tissue at the recipient site, donor site morbidity and cosmetic concerns, need for another surgical procedure and the time-consuming nature of the second procedure5. For allografts, these limitations are the a-vailability of a healthy graft, risk of i-nfection and transmission of fatal vi-ral diseases, inefficiency of the graft to incorporate with the healing of the recipient site, rejection of the graft, r-e-injury due to the low biomecha-nical performance of the allografts and ethical concerns2,6,7. Also,

the presence of many unknown zoonotic diseases in animals, about which we have no information, could be another major concern6,8,9.

For the above reasons, tissue engi-neering has been introduced to reduce these limitations and improve the outcome of incorporation of the tissue-engineered grafts and improve the healing processes of injured ten-dons5,10,11. In the last decade, tissue engineering has been improved and much advancement has been achieved12,13. Several types of scaffolds with different technologies have been introduced so that nowadays there are many commercially available tissue-engineered products in the market13. However, most of these tissue-engineered products have not passed in vivo tests and most of the tissue-engineered researches have mainly focused on in vitro assays6,12,14. In addition, in vivo studies regarding the role of tissue-engineered prod-ucts on regenerative medicine have not focused on mechanistic researches, and they have only observed the quali-tative results with poor subjective data5,6. Therefore, there are many controversies between the results of the in vivo studies and there are also many differences between the sources, designing and preparation methods of the tissue-engineered grafts, which make their comparison hard to fol-low5,7,15,16. Regardless of the efficacy of these tissue-engineered products, there are some great concerns that should be addressed in future studies13.

This study was aimed to introduce the role of tissue engineering in ten-don reconstructive surgery and regen-erative medicine, and has focused on

* Corresponding authorEmail: dr.ali.moshiri@gmail.com1 Division of Surgery, Department of Clinical

Sciences, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

2 Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

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Critical review

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F�� �������� ��������: Moshiri A, Oryan A. Role of tissue engineering in tendon reconstructive surgery and regenerative medicine: current concepts, approaches and concerns. Hard Tissue. 2012 Dec 29;1(2):11.

the general guidelines for those investigations that translate basic to clinical researches in the field of tendon tissue engineering.

Classification of tendon injuriesTo design a suitable treatment strat-egy for tendon reconstruction, it is i-mportant to know the nature of the tendon injury and its correlation wi-th the goals of tissue engineering1. Tendon injuries are varied between degenerative tendinopathies and te-ndon defects2,17 and could be group-ed under three classifications2. The first classification is the sharp ruptu-red/transected tendon injuries18. T-hese types of tendon injuries happen due to extrinsic traumatic forces su-ch as a gunshot or vehicular trauma and/or during orthopaedic procedu-res when the tendon should be incis-ed to extend the surgical approaches or when the bone fixative materials are implanted to stabilize fractured bones1. In this type, the tendon ends are sharply transected and direct su-turing is possible19. In the second ty-pe, classified as the blunt tendon ru-ptures, the injuries have resulted fr-om high stress forces such as those resulting from sport injuries. Degen-erative tendinopathic conditions are other subtypes of these injuries17. T-hese types of injuries are divided in-to three subgroups including stress, strain and rupture injuries3. Based o-n the nature of the injuries from par-tial to complete tear of all the collag-en fibres, these types face different challenges3. In complete blunt tend-on ruptures, the edges of the ruptur-ed tendon are not uniform and are f-uzzy; thus, it is necessary to debride these tendon edges and facilitate su-ture placement. Accordingly, some parts of the ruptured tendon may have been lost and direct suturing is of limited value in this condition2,3,9. Usually, the defect area is not large and healing response is not the maj-or concern in these types of injurie-s4,20. The last classification belongs to those injuries that have resulted

from massive trauma, burning and s-evere degenerative changes, which r-esult in the formation of large defe-cts in the injured tendons. In these t-ypes of injuries, healing response of t-he injured tendon is of great concer-n3,4,9.

Process of tendon healingTendon healing could be classically divided into three phases including i-nflammatory or exudative, fibroplas-ia or proliferation and remodelling p-hases. There is some overlapping be-tween these phases21 and there are other transitional stages in each pha-se, which have never been clearly de-fined to date17. Classic tendon heali-ng could be expected to happen in th-ose tendon injuries with low degrees of tissue loss, but characterization of tendon healing could differ based on the severity of tendon injury and the treatment modality22. For example, t-he healing characteristics of tendon defects treated with autografts have some differences when compared w-ith those treated with allografts. Per-haps these differences are larger wh-en the tissue-engineered grafts are used to repair the defect site12,17,23,24. Inflammatory phase of tendon healingInflammation is a major start key of tendon healing, without which effect-ive healing is not expected24.

Lag stageImmediately after tendon injury, the lag phase starts. The vascularity of t-he injured tendon is impaired so that ischaemia commences in the injured area17. The tenocytes die and deliver the cytokines and proteases. This co-uld be due to lysis of the cell membr-anes21. These cytokines increase the permeability of the unsevered vascu-lar structures of the injured area17. In addition, injuries may result in vasc-ular rupture so that the blood supply enters the injured area23. By exposu-re of the platelets to the injured collagen fibres, they aggregate

with fibrin strands to form blood clot17. Platelets then deliver cytokines, growth factors (e.g. platelet-derived growth factor) and inflammatory medi-ators to initiate the inflammatory stage of the wound healing in the injured area23. By these mechanisms, some reversible changes take place in the endothelial lining of small venules and capillaries and the inflammatory cells including neutrophils, and then macrophages, lymphocytes and plasma cells enter the injured area24. The fibrin clot acts as both a chemotactic medium and scaffold for the inflammatory cells so that they can migrate on the fibrin strands throughout the injured area23.

Infiltration and debridingAt this stage (Fig. 1A), the inflammatory cells infiltrate the injured area and start to degrade the necrotic tissues and fibrin clot by phagocytosis and enzymatic lyses23,25. Neutrophils enter faster than other inflammatory cells (Fig. 1A) and play a major role in the presence of bacterial infection, for phagocytic activity and enzymatic lyses of debris, but do not play a crucial role in the following processes of wound healing1. Macrophages enter the injured area later than neutrophils and have significant roles not only in phagocytosis and proteolysis of the necrotic tissue, fibrin, degraded col-lagen and elastin fibrils and foreign body material but they also deliver matrix metallo proteinases (MMPs), cytokines, growth factors and angio-genic mediators in the injured area and have a crucial role in further heal-ing processes (Fig. 3A)17,23. MMP-9 and MMP-13 degrade the necrotic tissues and are useful in the inflammatory stage, and MMP-2, MMP-3 and MMP-14 change the inflammatory phase to fibroplasia and collaborate in tendon remodelling at later stages21. Cytokines and other chemo-attractant media-tors of macrophages chemotactically attract the mesenchymal cells, teno-blasts and endothelial cells into the injured area and by delivering growth factors, induce cell differentiation

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F�� �������� ��������: Moshiri A, Oryan A. Role of tissue engineering in tendon reconstructive surgery and regenerative medicine: current concepts, approaches and concerns. Hard Tissue. 2012 Dec 29;1(2):11.

and proliferation24,25. Therefore, mac-rophages have a major role during different phases of tendon healing26,27. Growth factors such as vascular endothelial growth factors (VEGF), basic fibroblast growth factors, plate-let-derived growth factors and tissue growth factor-β have significant roles in cell differentiation, cell prolifera-tion and tissue maturation and collaborate in all phases of tendon healing with different mechanisms19.

Fibroplasia phaseIn a routine tendon injury, fibroblasts are the dominant cells about five days post-injury and this gradually chang-es the inflammatory phase to fibropla-sia or proliferative stage. This stage can be divided into three sub-stages including early fibroplasia or stage of fibrous response, mid-fibroplasia or granulation tissue stage and late fibroplasia or amorphous collagenous stage18,28.

Fibrous responseIn this stage, the peritendinous fibro-blasts migrate from the injured tendon Sheath to the injured area29. This is the extrinsic mechanism of tendon heal-ing17,25. Intra-tendinous tenoblasts also infiltrate the injured area from the uninjured parts of the severed tendon1. This is the intrinsic mechanism of ten-don healing21. The function of tenocytes may vary based on their origin. Cells from the tendon sheath produce less collagen and glycosaminoglycans than epitenon and endotenon cells. How-ever, fibroblasts from the flexor tendon sheath proliferate more rapidly17. Intrinsic healing results in a better biomechanical performance and fewer complications; in particular, a normal gliding mechanism within the tendon sheath is preserved. In extrinsic healing, scar tissue results in adhesion forma-tion, which disrupts tendon gliding29.

The undifferentiated mesenchy-mal cells also migrate from the

peritendinous tissues and fill the injured area30. These cells are activated and differentiated into fibroblasts and endothelial cells by the local growth factors19. The final part of this stage is characterized by the proliferation of fibroblasts and endothelial cells23.

Granulation tissue stageTendon injury is associated with vas-cular damage, diminished blood supply and hypoxia, and the main conse-quences of hypoxia are further cell degeneration and necrosis23. Hypoxia is the key element which activates macrophages to release angiogenic factors and initiate angiogenesis in the injured area17 and VEGF has a major role in this regard19. The endothelial cells aggregate in the granulation tissue, proliferate and regenerate blood vessels (Fig. 1B)24,25. The newly formed vessels are pri-marily obstructed, but some of them connect to each other, re-canalize and some of them are able to be connected to the main circulation, but many of them die because of insufficient nutrition and hypoxia17. At this time, there is a reduction in the population of the neutrophils and the acute inflammation changes to chronic inflammation, in which macrophages, lymphocytes and plasma cells are the major inflammatory cells22,27,28.

Proliferating fibroblasts produce fibronectin, collagen, elastin and pol-ysulphated glycosaminoglycans such as hyaluronic acid, dermatan sulphate, chondroitin sulphate, heparan sulphate, heparin and glucosamine23,30. It has been shown that glycosaminoglycans have important roles in tendon healing, and they act as a scaffold for collagen deposition and collaborate in collagen fibril formation and differen-tiation23,25,28. Hyaluronic acid has also been shown to have a significant role in subsiding the inflammatory stage of wound healing23. Glycosaminoglycans and collagen type III, which are depos-ited by immature and mature fibro-blast, form the building block of the initial matrix architecture of the

Figure 1: Histopathological changes in tendon healing. (A) Inflammatory stage is characterised by the presence of numerous inflammatory cells in the injured area. (B) Early to mid-fibroplasia stage of tendon healing. The tissue is hypercellular, but insufficient amount of collagen has been produced by the tenoblasts. The healing tissue is highly amorphous. (C) Late fibroplasia. The collagen mass density has increased. The blood vessels have large calibre and are mature but the tissue is amorphous. (D)Early remodelling. The tissue is well aligned compared to that of (C). The cellularity has decreased and most of the blood vessels have been degenerated. (E) Maturation to consolidation stage of remodelling. The collagen fibres are highly mature and the cellularity has decreased compared to (E). (F) Normal tendon (H&E, Scale bar A–F: 120 µm).

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newly regenerated tissue. At this stage, the healing tissue has low echogenicity and homogenicity at ultra-sonographical examination, uniformly small-sized unimodal col-lagen fibrils at the ultra-structural level, minimum ultimate strength, yield strength, maximum stress and modulus of elasticity at biomechani-cal testing (Figs 1B, 2A and 3B).1

Amorphous collagenous stage Fibroblasts (tenoblasts) mainly start to produce large amounts of type III collagen fibrils17; however, the colla-gen/glycosaminoglycans ratio, which is called collagen density, is low at this stage23,25. The metabolic activity of the tenoblasts is high19, the propor-tion of nucleus/cytoplasm is elevated and the transverse diameter of the newly regenerated collagen fibrils is still small, ranging from 32 to 64 nm19 (Fig. 3C). Such a granulation tissue is hyper-cellular, disorganized and highly vascular. The collagen fibres are hap-hazardly distributed and there is no correlation in the direction of teno-blasts, fibrous connective tissue and blood vessels at this stage. Therefore, glycosaminoglycan and collagen con-tent of the healing tissue gradually increase up to three weeks post-injury, but then they start to decline and reach their steady state at about five weeks post-injury (Fig. 2B)23,30.

Remodelling phaseThe remodelling or maturation phase of tendon healing can be divided into three different sub-stages, including early remodelling or alignment stage, mid-remodelling or maturation stage and late remodelling or consolidation stage1,17.

Alignment stageAt this stage, the inflammation has subsided and the amorphous tissue has filled the injured area so that the continuity of the injured tendon is established1. This provides an oppor-tunity for the patient to use their injured limb so that the weight-bearing

Figure 2: Scanning electron microscopy of different stages of tendon healing.(A) At early fibroplasia, a low density of highly immature collagen is seen. (B) At late fibroplasias, the collagen density has increased. (C) At the early remodelling stage, the collagen fibrils tend to assemble to collagen fibres. (D) At the maturation stage of remodelling, the collagen fibres are completely differentiated from aggregation of the matured collagen fibrils. (E) At the consolidation stage of the remodelling phase, the collagen fibres are denser and more aligned. (F) Scanning morphology of the intact collagen bundle with its highly dense and align collagen fibres.

Figure 3: Transmission electron microscopy of different stages of tendon healing. (A) Inflammatory stage: a macrophage is seen with its characteristic cytoplasm and enzymatic granules. The collagen fibrils of the implant are lysed and new fibrils are produced in the tissue. (B) Early fibroplasia. The density of the collagen fibrils is low and they are randomly distributed in different directions. (C) Late fibroplasia: the density of the collagen fibrils has increased and they are more aligned. They are all unimodal and have small diameter. (D) Early remodelling: the diameter and density of the collagen fibrils have increased and they are highly aligned so that they have been only sectioned transversely in one ultra-thin section. (E) Mid-remodelling stage: the collagen fibrils are bimodal so that some of them are in the range of 32–64 nm and others in the range of 65–103 nm. (F) Normal uninjured tendon. The collagen fibrils are highly aligned, they are multimodal and their diameters vary from 32 to 270 nm (scale bar A: 1.6 µm; B:256 nm; C–F: 192 nm).

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Critical review

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F�� �������� ��������: Moshiri A, Oryan A. Role of tissue engineering in tendon reconstructive surgery and regenerative medicine: current concepts, approaches and concerns. Hard Tissue. 2012 Dec 29;1(2):11.

capacity and physical activity of the affected limb increases and weight-bearing forces can be transmitted between the bone–tendon–muscle complex23,26. These stress forces align the collagen fibres and blood vessels along the stress line (Fig. 3D)19. Accord-ingly, cellularity and transverse diam-eter of the injured area decrease (Fig. 1D). At this stage, the density of the collagen fibrils increases and their alignment improve so that they are mainly oriented uni-directionally along the longitudinal axis of the ten-don (Fig. 3D)22,23. Accordingly, the ultra-sonographical homogenicity increases and some improvement in the bio-mechanical properties of the healing tissue is seen1.

Maturation stageDue to the decrease in the cellularity and hydration of the tissue and the presence of a large amount of aligned collagen fibrils, the metabolism of the new tissue decreases and the blood vessels degenerate and resorb (Fig. 2D)21,31. Most of the newly regen-erated blood vessels disappear and only a few blood vessels, having large calibres, can be seen1. The weight-bearing capacity and physical activity of the patient increases and the immature, but aligned collagen fibrils start to aggregate and differentiate into larger and more mature collagen fibrils (Fig. 3E)24,26. The polysulphated glycosaminoglycans (e.g. chondroitin sulphate, dermatan sulphate and ker-atin sulphate) collaborate in the mat-uration and differentiation of collagen fibrils30. This increases the transverse diameter of the highly aligned colla-gen fibrils so that their diameter increases from 32–64 to 65–103 nm (Fig. 3E)18. However, their diameter is still much smaller than that of the nor-mal tendon (e.g. 250 nm) (Fig. 3F)25,31. Upon maturation of the collagen fibrils, type III collagen of the healing tissue decreases and is replaced with type I collagen21,30. At this time, the tenoblasts mature completely and they transform into metabolically

inactive tenocytes that are histologi-cally characterized by longitudinal cigar-shaped nuclei with a much higher proportion of nucleus/cytoplasm than tenoblasts22. The above changes accel-erate the ultra-sonographical echogeni-city and biomechanical properties of the healing tissue so that in a normal uncomplicated healing more than 50% of the normal contralateral tendon’s characteristics should be achieved at this stage27.

Consolidation stageThis is the slowest stage of tendon h-ealing and could continue for years or even to the end of life17. At this st-age, the matured collagen fibrils agg-regate together and produce larger c-ollagen fibrils (>103 nm). There is al-so improvement in the quality of cro-ss-linking of these collagen fibrils. M-oreover, the collagen fibrils are aggr-egated and covered by endotenon, e-pitenon and paratenon so that the c-ollagen fibres, fibre bundles and fasc-icles are formed (Fig. 2D, E)23,26. Wh-en the fascicles are formed, it can be suggested that tendon healing is in i-ts final stage27. By this time, the bio-mechanical characteristics of the ne-w tendon are almost comparable to those of the normal tendon, but in a large injury it may never reach its n-ormal value17. At this stage, the histo-pathological, ultra-sonographical and ultra-structural morphologies of the repaired tendon are also inferior to t-hat of the intact tendon, and the reg-enerated tissue is still hypercellular, the diameter of the collagen fibrils is far behind that of the normal contral-ateral tendon, and the hierarchical o-rganization of the tendon from colla-gen fibril level to fascicle is not prop-erly developed yet (Fig. 1Evs.1F)18,28.

DiscussionThe authors have referenced some of their own studies in this review. The-se referenced studies have been con-ducted in accordance with the Decla-

ration of Helsinki (1964) and the protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed. Animal care was in accordance with the institution guidelines.

Limitations of tendon healing based on the type of tendon injuriesTendon healing is a complicated and targeted process but it has some limitations20,32. Generally, the major limitation of tendon healing is development of peritendinous adhesions18,22,29. Incidental injuries or surgical operations may result in disruption of the paratenon or tenosynovium. Also, if this structure is preserved, it usually lyses by the chemical activity of the MMPs and other degrading enzymes which are secreted during the inflammatory phase of tendon healing29. Lack of effective mechanisms to guide proliferation and direction of the fibroblasts and collagen fibres at the fibroplasia stage results in proliferation of fibroblasts in a haphazard fashion20,29,32.

Actually all types of tendon injuries are subjected to the development of peritendinous adhesions, but when tendon injury is more severe and the size of the defect area is larger, develop-ment of peritendinous adhesion is more aggressive and the outcome is much poor2,29. In such cases with sig-nificant tissue loss, there is also another limitation that would have a major role in the functional impairment24. When the tendon defect is larger than that to be directly repaired by primary surgical repair, the amount of periten-dinous adhesion substantially increases because there is no scaffold for the healing cells to guide them to prolifer-ate along the stress line of the tendon.Accordingly, these cells proliferate in different directions. In such large tendon defects, the healing capacity decreases because the fibroblasts migrate in the

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peritendinous fascia and hinder muscle insertion, resulting in muscle fibrosis. Therefore, the population of the migrated fibroblasts in the defect area is reduced, which is followed by a reduction in the amount of collagen production. Continuity of the defect area in such a tendon injury may not be established5,17. Therefore, large tendon deficits have more significant limita-tions, including the development of peritendinous adhesion, muscle atro-phy, muscle fibrosis and improper healing response29. These limitations should be considered when a proper tissue engineering approach is to be designed2.

Role of tissue engineering in tendon regenerative medicineIn the last decade, tissue engineering has seen much advancement and sev-eral manufacturing technologies and treatment modalities have been intro-duced to reduce limitations of tendon healing and to improve the healing response5,12,33. Basically, tissue engi-neering consists of three different parts, including scaffolds, stem cells and healing promotive factors5,34. A major advance ment in tendon tissue engineering is related to the scaffolds. The first step in tendon regenerative medicine is to design a suitable envi-ronment for cell migration, proliferation and a navigator for tissue alignment, remodelling and maturation35. There-fore, there are several factors that have an impact on the effectiveness of the scaffold in this regard including the molecule(s) from which the scaffold is manufactured (basic material of the scaffold), architecture of the scaffold, diameter and orientation of the fibres, their biological characteristics and the amount of free spaces and pore size5,16. There are also a numbers of other issues that should be considered in manufacturing a scaffold12; for exam-ple, a suitable scaffold for tendon tissue engineering should be cyto-compatible in vitro and biocompati-ble and biodegradable in vivo5,16,33,36,37.

Unfortunately, most of the exogenous-based biomaterials for tendon repair have serious limitations, such as lower capacity for inducing cell proliferation and differentiation (tenoinductivity), poor biocompatibility and remodel-ling potentials (tenoconductivity)16,33,38.To date, no manufactured scaffold has passed all the above issues both in vitro and in vivo and this is the greatest concern39. Here, we briefly discussed each of the above characteristics16.

Basic material of the scaffoldSeveral materials have been used so far to produce tissue-engineered scaf-folds; however, few of them have been effective in tendon tissue engineering and regenerative medicine2,40. Gener-ally, they can be divided into three major groups including biological (natural), synthetic and hybrid mate-rials12,41,42. Biological materials such as collagen, elastin, gelatin, chitosan, albumin, alginate, fibrin and chondroi-tin sulphate have been shown to be effective in tendon healing36,40,43,44. Actually, these materials are biocom-patible and biodegradable2. Their toxicity is low and has some benefi-cial biological role after implantation in the injured area41.

Mature tendons are composed of more than 90% type I collagen1. This molecule has an excellent physical property and in vivo activity, and its production is not expensive36. It can be easily formed into any shape and architecture, and is equipped with most of the healing promotive fac-tors36. Elastin is also present in ten-dons in a much less proportion (about 1%) and its major application in tis-sue engineering is to produce vascu-lar scaffolds12,17. Chitosan is a natural polysaccharide obtained from insects. Nowadays, this molecule is the focus of many research programmes, and it has been shown that this molecule has excellent biological activity5. All these materials are biodegradable and biocompatible2.

There are also some non-biodegradable biological materials such as silk and carbon fibres5. The usage of carbon fibre did not continue because of its high toxic effect and serious inflammatory reactions. How-ever, investigations into silk are still in progress, but most of the studies in this regard are in vitro investigations that have low value in translational medicine12,34. Synthetic materials such as polycaprolactone (absorbable), polydioxanone (absorbable), polyga-lactin 910 (absorbable) and nylon (non-absorbable) are other options with invaluable results42,45,46. Many researches have focused on the in vitro characteristics of such materials and those who investigated their in vivo efficacy have not suggested their clinical application and claim that they induce exaggerated inflammatory reactions, are highly cytotoxic and have poor outcome because of their high rates of rejection after implanta-tion2,12,41,46. Their major application is in vascular tissue engineering and body wall defects, and most of them are produced by petroleum material12,41,46. In fact, their first application in medi-cal sciences was in the surgical field as suture materials47. Their outcome was excellent compared to biological materials when their usage was limited to surgical sutures12. Probably, their merit as a surgical suture can be attrib-uted to their excellent biomechanical properties47. Also, these materials as surgical suture do not have consider-able toxicity because the amount of foreign material is considerably less than the scaffolds constructed from these materials47. Therefore, due to the excellent biomechanical and physical characteristics of these materials, they were never deleted from the field of tissue engineering, but their propor-tion in tissue-engineered scaffolds has greatly reduced46. Therefore, they have been combined with biological materials to decrease their limitations. However, in vivo studies

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regarding their efficacy in tendon regenerative medicine are rare12,46.

Dimensions of the scaffoldsRegardless of the different technolo-gies introduced, most of the tissue-engineered scaffolds designed for tendon tissue engineering are bidi-mensional14,48. These types of scaffolds are commercially available in the market and are produced as films, patches or membranes (Fig. 4A)2,12,48. Their major application in clinical practice is to inhibit the development of peritendinous adhesion during tendon healing (Fig. 4B)5. For this purpose, the injured area is surgically repaired by tension sutures and these scaffolds are wrapped around the injured area of the tendon with a mini-mum gap (Fig. 4C)9,48. In tendon injuries with significant tissue loss, as discussed in this article, the defect area should be repaired by grafts49. In this regard, tridimensional scaffolds are more suit-able (Fig. 4G, I and J)5,50. Unfortunately, tissue engineering is not capable of producing these types of scaffolds, and if produced, because of lack of in vivo studies, their effectiveness in tendon regenerative medicine is unclear12,49.

Architecture of the scaffoldsSeveral technologies have been intro-duced in this regard; however, tissue engineering is still in progress and it is expected that newer designs with more enhanced architectures would be manufactured in the near future5,49. The classic tissue-engineered prod-ucts are the allo- or xeno-geneic-based grafts that are processed only by acellularization technologies15,16,51,52. Thus, their architecture could not be re-designed in a more effective man-ner after implantation in the injured tendons15. Despite acellularization of such grafts, their rejection rate is high and therefore, they cannot be consid-ered as tissue grafts5,7.

Newer approaches such as gel sys-tems, porous systems and electro-spinning have been introduced2,45,53,54. Each of these technologies has its own purpose, but the gel system is used to produce a tridimensional envi-ronment for cell culturing purpos-es5,40,53. The in vivo application of the gel system has significant limitations, including a low biomechanical per-formance when used as a graft in the injured area33,50,55. Also, the absorp-tion rate is fast so that few days after

implantation, the architecture is completely depleted by the inflamma-tory cells and mediators2,12. Moreover, the orientation of their polymerized fibres is randomized and is not suita-ble for the purposes of tendon tissue engineering2,50,56.

Porous scaffolds are used to pro-duce both bi- and tridimensional scaf-folds with different purposes (Fig. 4H). Their major application is in bone and cartilage regenerative medicine12. By this technology, several pores were introduced in the implant with the aim of filling the newly regenerated tissue, after implantation. Due to the place-ment and randomized distribution of these pores at different sides of the scaffold, the orientation of the regen-erative tissue is not aligned; this irreg-ularity is not desirable in tendon tissue engineering as proper alignment of the newly regenerated tissue is a must (Fig. 4H)34. In these types of scaffolds, alignment of the newly regenerated tissue is not a major concern because of the nature of the tissue that should be reconstructed56. Electrospinning is another but highly expensive and time-consuming technological approach2,42,54. By this technology, it is possible to align the polymerised fibres of the scaffold (Fig. 4F)2,44. Also, the diameter of the polymerised fibres can be designed to produce different ranges of fibres from nanometric to micrometric scales54.

Other physical characteristics of the scaffoldsRegardless of the above factors that are necessary for consideration, there are some other issues that should be addressed2. For example, the diameter and orientation of the polymerized fibres of the scaffold are important (Fig. 4F)40. Both these characteristics can be controlled and ordered when the new design is approached40,44. The tendon-engineered scaffolds should have fibre diameters varying between nano-scale and micro-scale57,58. It has been shown that controlling the scaf-fold fibre diameter is critical in the

Figure 4: (A) Bidimensional scaffold has been produced by tissue engineering technology. (B,C) This scaffold has been wrapped around the injured area of tendon to reduce development of peritendinous adhesion. (D,E) Bidimensional scaffold has been used for augmentation of small tendon defects. (F) Scanning electron micrograph from the surface of the synthetic tissue engineered scaffold. (G) Synthetic tridimensional tissue-engineered scaffold for tendon and ligament repair. (H) Synthetic tridimensional porous scaffold for bone and cartilage repair. (I) Collagen-based tridimensional tissue-engineered scaffold has been used in repairing a large tendon defect. (J) Polydioxanone bidimensional scaffold has been wrapped around the tissue-engineered collagen-based bioimplant and has been implanted in a large tendon defect.

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design of scaffolds for functional and guided connective tissue repair, and provides new insights into the role of matrix parameters in guiding soft tissue healing57,58.

A tendon’s normal architecture is composed of collagen fibrils with nano-metric diameters and collagen fibres and fibre bundles with different micro-metric diameters.Therefore, a suitable scaffold for tendon tissue engineering should consist of both micro and nano-scale moderately to highly aligned fibres to be effective in guiding both the nano and microstructure of the newly regenerated tissue after implan-tation14,40,44. Pore size is another impor-tant factor40. Tendon is not similar to cartilage or bone architecturally and for this reason the size of the pores should be smaller than the scaffolds that are constructed for cartilage and bone tissue engineering35. In addi-tion, the number of pores should be fewer. Their orientation is also the most important factor2. Actually, in a well-designed tridimensional tendon scaffold, if there are six sides on the scaffold, only two sides should be porous in nature. Their position should be on the proximal and distal aspects of the implant just between the severed tendon edges. The other four sides at the periphery should be non-porous to reduce the amount of invasion of the peritendinous fibroblasts into the implant from the periphery.

Water uptake and water delivery of the scaffold is another important char-acteristic that should be addressed40. A well-designed implant should quickly absorb liquids but deliver them slowly56. These characteristics are mainly de-pendent on the above mentioned fac-tors44. With these characteristics, the implant is able to absorb the cellular structures and healing mediators in its architecture and maintain them for a long time56. These characteristics are also used for drug delivery44. The spe-cific growth promotive factors could be assembled within the scaffold and de-livered in a suitable time after implan-tation of the graft with the aim of

increasing the efficiency of the treat-ment modality2.

Biological characteristics of the scaffoldsBiological characteristics are a major concern in the field of tendon tissue engineering10,33. At least five possible biological responses have been suggested after implantation of extra cellular matrixes including (i) ECM non-incorporating responses: (a) encapsulation; (b) rejection; and (ii) ECM-incorporating responses: (a) resorption; (b) integration with progressive degradation; (c) adoption and adaptation7,8,37,44.

Several attempts have been made to improve the biological behaviour of the scaffolds with the aim of improv-ing their biocompatibility, biodegrada-bility and bioefficacy and decreasing the rejection rate7,39,44,51,59. Acellulariza-tion or decellularization is the first step in this issue, especially in those biologi-cally based tissue-engineered grafts obtained from allo- or xenografts7,9,33,51. Selecting a proper material is another approach34,52,60. For example, collagen has been shown to have excellent bio-degradability and biocompatibility2,51,61, on the other hand, synthetic materi-als have low biocompatibility12,33,44; thus, the amount of biologically based materials should be increased and the percentage of synthetic materials should be decreased when designing a hybrid scaffold. Sterilization is another factor that should be addressed2. By removing all cellular and microbio-logical structures, the immune response could be reduced in order to increase the chance of graft incorporation in the healing process15,33,34.

Increasing the number of specified molecules in the architecture of the scaffold could reduce the limitation of each molecule15,59. The best-suited scaffold for tendon tissue engineering should have the following biological behaviour44: it should not acutely or chronically be rejected by the host immune response and should not be encapsulated by fibrous connective

tissue44. But it is imperative to mildly initiate the immune reaction and modulate inflammation because this can increase the healing rate2,33. This incorporating behaviour should not be accompanied by acute resorption of the scaffold37. The best biological behaviour of a suitable scaffold is to be accepted as part of a new tendon. However, to date, this behaviour has not been shown for soft tissue scaf-folds, but by designing suitable modali-ties, it may be possible to preserve some parts of the scaffold in a manner that is acceptable as part of the new tendon33,35. Integration with progres-sive degradation is also well accepted because the graft incorporates in ten-don healing and collaborates in differ-ent stages of the healing process37.

Optimization of the scaffolds as the �inal stepOther aspects of tissue engineering have been developed together with the development of scaffolds including stem cells and healing promotive fac-tors13,62. A scaffold provides a suitable substrate for cell attachment, cell proliferation, differentiated function and cell migration. Scaffold matrices can be used to achieve drug delivery with high loading and efficiency to specific sites43. Several investigations equipped tissue-engineered scaffolds with different types of stem cells and healing promotive factors to increase the efficiency of tissue-engineered grafts13,33,43. The transplanted cellular structures have been shown to signif-icantly improve the healing quality of the repaired tissue63. There are also many researches that have shown the efficacy of different healing promotive factors33,60. By equipping the tissue-engineered scaffolds with different types of healing promotive factors, the healing response can be controlled, there by making it more efficient in increasing the quality of the repaired tissue33. Glycosaminoglycans have been the main focus in this regard56. Hyalu-ronic acid is one of these agents23. This glycosaminoglycan has been shown

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to modulate the inflammatory phase of tendon healing and increases the rate of healing process18,23. It also increases the diameter of the newly regenerated collagen fibrils and improves the alignment of the newly regenerated tendon, in vivo23. Growth factors are another option17,33,60; for example, basic fibroblast growth fac-tor has been shown to have an impact on the cell proliferation, maturation, collagen production and remodelling phase of tendon healing1,19,24.

Tarantula cubensis is another novel agent that has been shown to decrease the necrotic tissues in the injured area, modulate the inflammatory reac-tion and reduce the amount of peri-tendinous adhesions22,28. Platelet-rich plasma is another healing promotive agent that initiates cell proliferation and tissue maturation20,23,32. These beneficial effects have been suggested to be due to the growth factors that are delivered from the platelets62,64. It seems that by equipping the tissue-engineered scaffolds with cells and healing promotive agents, the tissue-engineered graft could be optimized to open new insights in the field of tendon reconstructive surgery and regenerative medicine12.

ConcernsAs it has been stated in this article, most of the researches in the area of tendon tissue engineering have not focused on the in vivo conditions and for this reason the real efficacy of such tissue-engineered products and their biological behaviour is unclear. Such concern particularly increases when these types of tissue-engineered prod-ucts are introduced as tissue substi-tutes in clinics without passing the scientific requirements and approval12. In addition, the significance of in vitro studies in translational medicine is also questionable. For example, it is not clear as to how the cultured stem cells on tissue-engineered scaffolds would be effective when the host–scaffold interaction and immune response to implantation of the scaffold is not

clear and not defined. It is not clear how these cells remain alive in the inflammatory phase of tendon heal-ing when large amounts of degrading enzymes are delivered by the inflam-matory cells. Therefore, future inves-tigations should consider the in vivo tests and the in vivo researches should focus on the mechanistic approaches and not just on the observations of the final outcome. This includes design-ing several observational time points after implantation of tissue-engineered grafts using different observational methods to comprehensively discuss about the mechanism of the host-immune response to the graft implan-tation and host toleration to the for-eign body exposure. For observation of the final outcome, it is also suggested to test the repaired tissue with differ-ent methodologies including both physical and chemical characteristics of the repaired tissue together with the morphological analysis at diffe rent levels (e.g. histopathology, ultra-structure)65,66.

ConclusionDespite significant advancement in tissue engineering technologies and producing many different tissue-engineered products, tendon tissue engineering is still in its infancy and translation of the present investiga-tions to clinics is accompanied by several important concerns including graft healing incorporation, host immune reaction and in vivo efficacy of the tissue-engineered graft. There are several blind points in this regard that should be addressed. Tissue engineering is going to be applicable in the near future; however, we should understand what the right purpose is before designing the new approaches. Knowledge about the nature of ten-don injuries, healing process and tissue engineering is needed when tissue engineering is selected as the alterna-tive approach. In addition, the research-er should have enough knowledge of the normal structural hierarchy, func-tional, biochemical and mechanical

performance of the specific normal tendon to be able to simulate an appropriate applicable scaffold.

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