Advances in injured tendon engineering with emphasis on the role of collagen implants

Introduction Tendon injuries represent a significant clinical challenge to orthopaedic surgeons and investigators. Recently, many investigators claimed the beneficial effects of tissue engineering technologies in full-thickness tendon and ligament injuries both in vivo and in vitro. In this regard, several fabrication technologies with various types of biomaterials have been specially developed in recent years. In the present review, designing, fabrication and application of tissue-engineered products accentuating the role of collagen bioimplants in tendon tissue engineering is discussed. Conclusion Tissue engineering methods have not been able to differentiate the unimodal small-sized newly generated collagen fibrils from those of multi-modally differentiated fibrillar sizes of various diameters that are present in the normal tendons of a mature animal.


Introduction
Tendons have poor spontaneous regenerative capability, and complete regeneration after injury is not achieved despite intensive remodelling [1][2][3][4][5][6] .Tendon injuries are variable and could be divided into acute and chronic injuries 1 , and perhaps, chronic injuries are more difficult to cure 7,8 .Tendon injuries could be categorized by etiology too 1,7 .
The most important tendon injuries include sharp tendon ruptures, spontaneous or tendinopathic forms of tendon ruptures and those which are congenital or acquired deficiencies with significant defects and with a characteristic of no healing response 1,5 .Tendon ruptures are initially anastomosed by primary suture techniques; however, if a part of the tendon proper has been lost and the defect area is large enough, the elongation and transplantation techniques could be helpful 5,6,9 .
In those tendon injuries with large tendon gap, routine clinical techniques are not applicable, and the healing response of the repaired tendons is not promising and often recurrence of injury would be expected because of the inferior biomechanical performance of the healed tissue 1 .Oryan and Moshiri 7 demonstrated that the ultrastructural diameter and modality of the collagen fibrils are correlated with the biomechanical performances and there is a significant gap between the collagen fibril diameter of the repaired and intact tendons even in the long term after tendon injury 4,7 .The Achilles tendon is the most frequently ruptured tendon 10 .It has been reported that the rate of re-rupture of the repaired Achilles tendons in the young and athletic populations and in the chronic large and massive rotator cuff tears remains high despite improvement in surgical techniques, suture design and postsurgical management 6,9 .Both acute and chronic tendon ruptures can dramatically affect a patient's quality of life and require a prolonged period of recovery before returning to pre-injury activity levels 10 .
A surgeon has several options to choose a graft for tendon reconstruction.
The autografts have several advantages, as they are not involved in the transmission of diseases, do not initiate host's immune reaction and are inexpensive 9 .However, the autografts have some serious limitations for tendon replacement, including limited availability, increased operative time, adverse functional changes including muscle weakness at the donor site and graft site morbidity, which may have a detrimental effect on the final outcome of the surgical reconstruction 9 .The allograft tendons offer the advantage of no donor site morbidity; however, limitation of their supply together with their high cost is prohibitive.There is concern for disease transmission as well as long-term viability.Allograft tissue carries the risk of transmitting bacterial and viral diseases, including human immunodeficiency virus and hepatitis.While the risk of transmission is low, it must always be considered as a possible complication 11 .Synthetic graft materials such as nylon, carbon fibre, silicone and Dacron have been used with limited success.These materials fail to offer the adaptability and flexibility seen with native tissues, which are capable of undergoing extensive remodelling 9,12 .More specifically, complications such as exaggerated inflammatory response, antigenic reaction, fixation site failure and long-term biocompatibility problems are seen with the synthetic grafts 9 .
Tissue engineering is a multidisciplinary approach that aims to induce repair, replacement or regeneration of tissues or organs 9 .Many strategies have recently been developed by tissue engineering technologies with the hope to solve these problems 2,[13][14][15][16] .A collagenous material as an excellent Licensee OA Publishing London 2012.Creative Commons Attribution License (CC-BY) Competing interests: none declared.Conflict of interests: none declared.
All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.
All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.For citation purposes: Oryan A, Moshiri A, Sharifi P. Advances in injured tendon engineering with emphasis on the role of collagen implants.Hard Tissue.2012 Dec 29;1(2):12.biological and biocompatible biomaterial has been lionised by many investigators and used to fill the defect area of the ruptured tendons and ligaments both under in vitro and in vivo conditions resulting in promising effects on tissue regeneration and physical performance of the injured tendons [17][18][19][20][21][22] .Many different collagen structures with a variety of curative effects have been used with or without a combination of some other bioactive molecules or stem cells 6,[23][24][25][26][27] .
Therefore, the aim of the present study was to review the most recent investigations on the effects of tissueengineered biocompatible and biodegradable collagen-based scaffolds/ implants on tendon healing.In addition, the most significant methods developed in tissue engineered collagen-based scaffolds/implants for tendon/ligament repair are introduced in this review.The blind points of previous studies and future areas of research have been magnified.

Role of collagen in tendon engineering
When musculoskeletal tissue is lost due to injury and/or illness, the defect is generally filled with natural tissue because artificial materials have problems of bioaffinity 9 .However, natural tissues also have supply and infection problems.If a biologically based artificial material has the same biological properties, it can replace the natural tissue for grafting 28,29 .Collagen is recommended by many investigators as an excellent biological material and it can be formed in a manner to produce a highly biocompatible and biodegradable artificial graft 9,25,[30][31][32] .
There are several commercially available collagen-based grafts that are derived from a variety of allogeneic (dermis) and xenogeneic [dermis and small intestinal sub-mucosa (SIS)] sources.An extensive amount of basic science and pre-clinical models have demonstrated that an extra-cellular matrix (ECM) patch produced from collagen materials may offer improved healing rates with a biomechanical profile that nearly reproduces the characteristics of the native tendon.This is the art of tissue engineering technology 9,25,32 .Porcine dermal (PD) collagen has been used for reinforcement of several human body tissues with success and has been shown to act as a durable, permanent tissue scaffold that assists healing 20,27,32 .Use of PD collagen as an augmentation graft in treatment of massive rotator cuff tears has been shown to be safe, providing excellent pain relief with a moderate improvement in active ranges of motion and strength and in most patients, and is associated with improved clinical outcome 32 .Lee 31 demonstrated that using an acellular human dermal tissue matrix to augment Achilles tendon primary repair in neglected Achilles tendon ruptures offered desirable outcomes without loss of function or additional surgical exposures observed with traditional flap, autograft or tendon transfer procedures.A similar investigation from a series of 9 consecutive cases of Modified Bröstrom stabilization with OrthADAPT™ Bioimplant augmentation with 9 months of follow-up has been reported by Fridman et al. 33 Their results indicated that the OrthADAPT™ Biologic Collagen provided support for augmentation and enhanced the stability of Modified Bröstrom procedure.Additionally, it prevented the need for tendon transfer and was free of its inherent complications.
Many investigators have selected collagen molecules to produce collagenbased, two-and three-dimensional (3D) scaffolds in different gels and nanostructure systems, aiming to improve tendon healing with the hope of future usage in the clinical cases associated with large tendon defects 14,15,[34][35][36] .The knowledge of design, fabrication and production techniques with the exper imental and clinical results would be helpful for orthopaedic surgeons to select and use the best biomaterial options and improve the structural and physical properties of the injured tendon.These criteria could be expanded to all other synthetic and biological scaffolds.Here the authors discuss the different approaches of fabrication and some of the most important in vitro, in vivo and clinical researches in the field of collagen bioimplants.

Collagen biocompatibility and degradation
Acellular collagen materials intended for biomaterial and tissue engineering usage could be prepared by selective and controlled hydrolysis of carboxyamides from asparagine and glutamine residues of type I collagen present in the pericardium, tendon and intestinal submucosa, mostly from bovine and less from other animals.This procedure has been analysed and recommended for the preparation of collagen biomaterials with variable physicochemical properties and macromolecular arrangement with respect to fibril formation and with potential use in tissue engineering 37 .The most significant reason in attracting the researches to collagen-based scaffolds is related to collagen biocompatibility and its unique degradation behaviour 30 .Buchaim et al. 14  All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.
All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.For citation purposes: Oryan A, Moshiri A, Sharifi P. Advances in injured tendon engineering with emphasis on the role of collagen implants.Hard Tissue.2012 Dec 29;1(2):12.matrix following implantation in the surgically created bone defects of the neoformed osseous tissue in rats.Their results demonstrated that the implanted collagen matrices were highly biocompatible and acted as a scaffold in inducing bone formation.The biological activity of the scaffolds used in tissue engineering applications has been well approved and hypothetically depends on the density of available ligands, and the site of the scaffold at which specific cell binding occurs.Therefore, designing a proper collagen-based scaffold could alter the collagen biocompatibility in a manner to make a new revolution in tissue regenerative medicine 9,38 .
Despite using the collagen-derived scaffolds in the regenerative medicine, little is known about the degradation mechanisms of these scaffolds in vivo.The goal of tissue engineering technologies is to improve the biocompatibility and absorption rate of different types of scaffolds, aiming to accelerate the regeneration of new tissues in the defects 9,14,39 .It has been shown that various materials used in tissue engineering technologies have different absorption rate and mechanisms of actions, and these qualities would be useful for drug delivery and repair outcome when the scaffold is going to be fabricated.When non-cross-linked dermal sheep (NCDS) and gelatin discs were implanted subcutaneously in mice by Ye et al. 40 despite the presence of a high number of macrophages and influx of neutrophils in the NCDS discs, they showed a very low degradation rate.This was shown to be attributed to the presence of the matrix metalloproteinase inhibitor TIMP-1.In contrast, the gelatin discs degraded quickly, due to efficient formation of giant cells, presence of MMP-13 and absence of the inhibitor TIMP-1.While the DDR-2 receptor was not expressed in the gelatin discs, Endo180 and MT1-MMP were expressed, but at most times no co-expression was seen 40 .
Hao et al. 22 fabricated a biomimetic construct based on a combination of rabbit adipose-derived stem cells encapsulated in collagen I gel with different types of polyglycolic acid (PGA) scaffolds and implanted it into a 15-mm length critical-sized segmental radial defect.Their results showed that the rate of degradation of the scaffold was correlated with repair ability and regeneration 22 .By tissue engineering technologies, the biocompatibility and degradation rate of the bioimplants that is the purpose of application of the scaffolds could be modulated 9 .

Acellularization of the collagen-based tissues
Acellularization is one of the primary requirements to reduce the DNA and antigenic activity of the cellular elements of the scaffold in order to decrease the amount and severity of tissue reaction and host immune response 20,31 .In the autogeneic and allogeneic transplantation of the tissue, there are some degrees of tissue reaction and tolerable host immune response after implantation of the graft 9,11 .Most biological materials used in tissue engineering technologies, such as collagen-based structures, are provided from animal tissues such as the dermis, subcutaneous area, submucosa of the intestine, tendon and other connective tissues containing cellular elements 27,[41][42][43] .Direct implantation of these tissues to an in vivo model amplifies the host immune response and is sometimes associated with rejection 41 .Therefore, several investigations have developed acellularization technologies in tissue engineering to solve this problem.The morphology and molecular composition of ECM, including the basement membrane, is variable depending upon the organ from which ECM is harvested and the methods by which it is processed 39 .The porcine SIS has been recommended as a cell-free, biocompatible biomaterial for repair of the rotator cuff tendon tear 39 .Zheng et al. 41 have shown that one of these commercially available bioimplants, the Restore™ orthobiologic, implant, resulted in an inflammatory reaction characterized by massive lymphocyte infiltration.They demonstrated that Restore™ SIS is not an acellular collagenous matrix, and contains porcine DNA.On the other hand, the most popular commercially available acellular collagen scaffolds for tendon repair (Tissue Mend, Graft Jacket, etc.) have significant curative effects in those clinical and experimental cases associated with tendon defects.Due to their acellular structures, they resulted in minimal inflammatory reactions 6,20,26,42 .
A technique of tendon repair that is a combination of tendon graft inlay with a Pulvertaft-type tendon weave, using acellular human dermal matrix graft with resultant 'strip and shoelace' was developed by Branch 42 .He showed that the acellular human dermal matrix graft could be an optimal tendon repair material because it has low rejection potential, provides a scaffold effect for ingrowth of the healing tendon and maintains significant strength during the tendon healing process.
Sarrafian et al. 27 evaluated the effects of a cross-linked acellular porcine dermal patch (APD), and platelet-rich plasma fibrin matrix (PRPFM) in repair of acute Achilles tendon rupture in a sheep model and supported usage of APD, alone or with PRPFM, to augment Achilles tendon repair.In a similar study, Nicholson et al. 20 evaluated two commercially available rotator cuff repair augmentation patches in a sheep model.They created bilateral infraspinatus tears and repaired them with a cross-linked acellular PD patch (Zimmer Collagen Repair Patch), and a porcine SIS patch (Restore Orthobiologic Soft Tissue Implant; De Puy Orthopaedics).By 24 weeks, the chronic inflammatory response around the PD material had been largely replaced by normal inter-digitating connective tissue incorporation and the material was vascularized, infiltrated throughout its matrix by fibroblasts and was capable of allowing the ingrown cells and tissue to respond to different Licensee OA Publishing London 2012.Creative Commons Attribution License (CC-BY) Competing interests: none declared.Conflict of interests: none declared.
All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.
All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.For citation purposes: Oryan A, Moshiri A, Sharifi P. Advances in injured tendon engineering with emphasis on the role of collagen implants.Hard Tissue.2012 Dec 29;1(2):12.
degrees of mechanical forces.Finally, the PD patch performed its reinforcement role, did not produce a disorganized diversity of tissue, and did not appear to rarify or weaken with time.At 3 weeks post-operative, the fibrinogen levels were elevated in the animals implanted with SIS patches but not in the animals implanted with PD patches.At 9 weeks, while majority of the SIS patches appeared to have been degraded, lysed and absorbed, the PD patches were intact but not fully integrated with the surrounding tendon tissues.At 24 weeks, the PD patches were integrated into adjacent tendon tissues.Ectopic bone formation was observed in some tendons repaired with SIS patches.Surprisingly, necrosis and rarefaction of the surrounding tissue matrix were observed around the non-resorbable suture in the shoulders repaired with suture alone (without any patch reinforcement) 20 .
Although most of the studies in this area used primary evaluation techniques limited to histopathology and biomechanical testing and their sample population was low for statistical analysis, it was substantial to approve acellularization as the first step to expand tissue engineering to clinical trials.
Presence and application of these tissue-engineered products is debtor to researchers, who have developed different types of acellularization technologies.Brown et al. 39 developed acellularization of the basement membrane of different tissues.They showed that the basement membrane structures can modulate cell growth patterns in vitro and the procedure was applied to delaminate, decellularize, disinfect and terminally sterilize the ECM scaffolds that did not appear to affect the integrity of the surface basement membrane 39 .A simple process combining acellularization and chemical oxidation to acellularize and modify the dense micro-architecture of the tendon in order to increase the porosity and pore size of the scaffold in comparison with the native tendon was employed by Whitlock et al. 43 They produced an acellularized/oxidized collagen scaffold from the flexor digitorum profundus derived from the Leghorn chicken feet.To assess the cell infiltration rate and short-term inflammatory response to the scaffolds in vivo, they implanted 1-mm thick 2 × 2-mm sized acellularized/oxidized tendon scaffolds which were sterilised with ethylene oxide subcutaneously in 10-week-old female CD1 mice.The implants were then harvested at 3, 7, 14 and 21 days post-implantation.They demonstrated that the cellular material, especially those of the nuclear material, can be removed from a dense tissue such as the digitorum profundusderived tendon of the Leghorn chicken, and removing the cellular material may reduce the potential for inflammation and disease transmission upon implantation of the scaffold.The digitorum profundus-derived tendon scaffolds did not exhibit histological or gross evidence of inflammatory reaction at 3, 7, 14 or 21 days when implanted subcutaneously in the immunecompetent mice.However, homogenous cell infiltration by mononuclear, elongated, fibroblast-like host cells was observed at the periphery as well as within the inner matrix of the implanted scaffolds at all time points 43 .
Zhang et al. 44 tried different methods of acellularization to produce tissueengineered intra-synovial flexor tendon construct with the use of an acellularized flexor tendon scaffold re-populated with intra-synovial tendon cells.The intra-synovial flexor tendons of rabbits were acellularized by high concentration NaCl+SDS, Trypsin/EDTA, Trypsin/ EDTA+Triton X-100, Triton X-100, Triton X-100+SDS, and freezing at −70 °C followed by application of Trypsin/ EDTA+Triton X-100.The epitenon and the endotenon cells were also isolated from the intra-synovial tendons of rabbit and expanded in culture.The acellularized tendon scaffolds were then reseeded with these cells.An optimal acellularization was achieved by freezing at −70 °C followed by trypsin/EDTA+Triton X-100 44 .
Although there are many different techniques to acellularize the tissueengineered collagen scaffolds as the first step in tissue engineering technology, all of them have similar goals, aiming to decellularize collagen implants to reduce host immune reaction and improve repair outcome.

Porosity of the implants
Porosity of the tissue-engineered bioimplant is another determinative quality, influencing the cell-scaffold interaction, distribution and viability.Collagen sponge (CS) was recommended as a suitable model of tissueengineered product because its high porosity makes it an excellent biocompatible architecture in vivo 45 .Generally, the inner 3D structure of the sponges influences the behaviour of cells.To investigate this influence, it is necessary to develop a process to produce sponges with a defined, adjustable and homogeneous pore structure.CS can be produced by freeze-drying of collagen suspensions.The pore structure of the freeze-dried sponges mirrors the ice-crystal morphology after freezing.The conventional freeze-drying process for fabricating glycosaminoglycan (GAG) scaffolds creates variable cooling rates throughout the scaffold during freezing, producing a heterogeneous matrix pore structure with a large variation in average pore diameter at different locations throughout the scaffold.Schoof et al. 45 developed the unidirectional solidification technique during the freezing process to produce CS with a homogeneous pore structure.It has been shown that by using this technique, the entire sample can be solidified under thermally constant freezing conditions.The ice-crystal morphology and size can be adjusted by varying the solute concentration in the collagen suspension.They have shown that CS with a diverse uniformity and defined pore structure can be produced 45 .O'Brien et al. 46 modified the process to produce more Licensee OA Publishing London 2012.Creative Commons Attribution License (CC-BY) Competing interests: none declared.Conflict of interests: none declared.
All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.
All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.For citation purposes: Oryan A, Moshiri A, Sharifi P. Advances in injured tendon engineering with emphasis on the role of collagen implants.Hard Tissue.2012 Dec 29;1(2):12.
homogeneous freezing by controlling the rate of freezing during fabrication to obtain more uniform contact between the pan containing the collagen GAG suspension and the freezing shelf by using smaller and less warped pans.The modified fabrication technique has allowed production of GAG scaffolds with a more homogeneous structure characterized by less variation in mean pore size throughout the scaffold compared to the original scaffolds 46 .
Colonization of the biodegradable polymer scaffolds with cell populations has been established as the foundation for engineering of a number of tissues, including cartilage, liver, bone and tendon.Within these scaffolds, cells encounter a porous environment in which they are able to migrate across the convoluted polymer surface to generate a homogenous cell distribution.Predicting the interactions between cells and pores is important if the scaffold's characteristics are to be optimized 47 .O'Brien et al. 38 with the aim of explaining the effect of pore size and specific surface area of the scaffold on cell viability and attachment produced GAG scaffolds with constant composition and solid volume fraction, but with four different pore sizes corresponding to four levels of specific surface area by the lyophilization technique.A significant difference in attachment of the viable cells was observed in the scaffolds with different mean pore sizes at 24 and 48 h after incubation.The fraction of viable cells attached to the GAG scaffold decreased with increasing mean pore size, increasing linearly with the specific surface area of the scaffold.The strong correlation between scaffold specific surface area and cell attachment indicated that cell attachment and viability are primarily influenced by the scaffold specific surface area of pore sizes for cells 38 .In a similar study on GAG scaffolds, in vitro, it has been shown that as the cell density increases, the force per cell to achieve a given strain in the scaffold is expected to decrease 16 .Salem et al. 42 studied the behaviour of fibroblasts and bovine aortic endothelial cells over a range of defined pore features.These pore features ranged from 5 to 90 µm in diameter and were fabricated by photolithographic techniques 47 .They reported that the behaviour of cells was dependent on the percentage cell coverage of the surface or density, pore size and cell type.Their results confirmed that within a tissue, engineering scaffold cells displaying fibroblast behaviour will tend to block pores even where the pore diameter is greater than the cell diameter 47 .
Biophysical properties of the twodimensional substrates have been shown to significantly influence cell migration.Elucidating the factors governing cell migration in a 3D environment is a relatively new avenue of research.Harley et al. 48investigated the effect of the 3D microstructure, especially the pore size and the Young's modulus, of GAG scaffolds on the migratory behaviour of individual mouse fibroblasts.They showed that the migration of fibroblasts, characterized by motile fraction as well as locomotion speed, decreases as the scaffold pore size increases from 90 to 150 µm 48 .Direct testing of the effects of varying Young's modulus on cell motility showed a biphasic relationship between cell speed and strut modulus and also indicated that mechanical factors were not responsible for the observed effect of scaffold pore size on cell motility 48 .

Fibre alignment
Processing scaffolds that mimic the ECM of natural target tissue in structure and chemical composition is a potential promising option for engineering physiologically functional tissue 2,49 .Cells must retain the ability to migrate into an adjacent template, or scaffold, after a ligament or tendon rupture.It has been shown that the cells in the human ACL retain their ability to migrate into an adjacent GAG scaffold in vitro, weeks after complete rupture 50 .
Cell migration plays a critical role in a wide variety of physiological and pathological phenomena as well as in the scaffold-based tissue engineering.Cell migration behaviour is known to be governed by biochemical stimuli and cellular interactions.The biophysical processes associated with interactions between the cell and its surrounding ECM may also play a significant role in regulating migration 3,48 .It has been shown that the most important biomechanical factor related to the migration speed of cells on the twodimensional scaffolds is stiffness 51 .Alignment of the collagen fibres is another important factor that contributes to cell migration behaviour.Several studies have demonstrated the effects of fibre alignment on cell/collagen behaviour 2,30,52,53 .Gigante et al. 2 evaluated the effect of fibre orientation on the cell viability and cytoskeletal organisation in an in vitro investigation.They seeded human dermal fibroblasts (HDFs) and tenocytes on two collagen membranes (CMs) and demonstrated that the multi-lamellar collagen I membrane with oriented fibres had better mechanical properties and afforded optimum cell proliferation and adhesion.The fibre arrangement of this scaffold provided an instructive pattern for cell growth and guided alignment of cells migrating from the ends of a crushed or frayed tendon to obtain a strong, correctly structured tendon, thus providing a viable clinical option for tendon repair 2 .Ifkovits et al. 53  All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.
All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.For citation purposes: Oryan A, Moshiri A, Sharifi P. Advances in injured tendon engineering with emphasis on the role of collagen implants.Hard Tissue.2012 Dec 29;1(2):12.results indicated that the scaffold's architecture and porosity are important considerations in controlling tissue formation 53 .
Many alignment techniques have been developed and applied in tissue engineering technologies.For several years, micro-grooved substrates have been evaluated as a means to orient cells in engineered tissues.Recently, Vernon et al. 54 fabricated thin (0.1-5.3 mm) planar and tubular CMs from the air-dried hydrogels of native, fibrillar type I collagen.They showed that the planar CMs supported robust attachment, spreading, and proliferation of HDFs and human umbilical artery smooth muscle cells.The collagen hydrogels were air-dried into microgrooved templates and subsequently removed in the form of grooved CMs with the potential to align cells.Transformation of the fibrillar collagen hydrogels into the grooved CMs with the capacity to support cell attachment and induce cell alignment may prove to be useful for a variety of applications in tissue engineering 54 .Kapoor et al. 55 have studied the role of microtopographical features on the cytomorphology, alignment, proliferation and gene expression of tenocytes.They used simple micro-fabrication approaches to create surfaces patterned with topographical features suitable for in vitro studies of tenocytes.These surfaces were composed of glass substrates patterned with polymeric ridges spaced 50-250 μm apart.Their results demonstrated that the microgrooves differentially impacted the tenocyte shape, alignment and matrix organization along the direction of the grooves.The groove widths significantly influenced the cellular alignment, with the 50 μm grooved patterns affecting alignment most substantially.Polarized light microscopy demonstrated that the mature collagen fibres were denser and more oriented within the 50 μm patterns.With regard to their results, it could be indicated that micro-topography affects cell density and alignment of tenocytes and leads to deposition of an aligned collagen matrix, but does not significantly impact the matrix gene expression or cell phenotype.These outcomes provide insights into the biology of tendon regeneration, thus providing guidance in the design of clinical procedures for tendon repair 55 .
Berry et al. 56 developed a collagen gel model to improve cell and matrix orientation.In their study, human neonatal dermal fibroblasts were seeded in type I collagen and the gels were cast in a racetrack-shaped mould containing a removable central island.Two of the models were mechanically stressed (20 and 10 mM), because complete contraction was prevented by the presence of the central island.The central island was removed in the third model (released) and the final model was cast in a Petri dish and detached, allowing full multi-axial contraction (SR).Cell viability was maintained in the 10-mM and SR models over a 6-day culture period but localized regions of cell death were evident in the 20-mM model.Cell and collagen alignment developed in the 20-and 10-mM models and to a lesser extent in the released model but were absent in the SR model.Cell proliferation and collagen synthesis was lower in the 20-mM model than in the other systems and there was evidence of enhanced matrix metalloproteinase production.The mechanical properties of the 20-mM model system were inferior to that of the 10-mM and released systems.The 10-mM model system induced a high level of cell and matrix orientation and presented one of the best options for tissue-engineered ligament repair involving an orientated fibroblastseeded collagen gel 56 .
Recently, electrospinning has been introduced as a unique and effective method of fibre orientation with some tremendous results.However, most of the studies on the electrospin collagen scaffolds are in vitro although the evidence and results are encouraging.Electrospinning has been optimized to produce non-woven, tissue-engineered scaffolds composed of individual fibrils less than 1000 nm in diameter.Electrospinning uses an electric field to process polymers into discreet fibrils.In this process, a polymer solution or melt is charged by a high voltage and is directed towards a grounded target.The electric potential drives, or pulls, the polymer solution across an air gap and the solvent carrier evaporates.Depending upon the reaction conditions, electrospinning can be used to produce fine particles of aerosol or non-woven matrices composed of sub-micron diameter fibrils.This size scale is far smaller than what can be achieved with conventional processing methods and approaches the diameter of collagen fibrils present within the native ECM 57 .From a material processing standpoint, electrospinning is rapid and efficient.Nanoscale fibrils can be deposited in a target mandrel in a dry, sterile state.This type of tissue engineering material can be expected to have an extended shelf life, which is an important consideration in the commercial distribution process.The material and chemical properties of an electrospun matrix can be regulated at several different sites.For example, the fibre diameter and average pore dimension can be controlled by regulating the concentration of materials present in the starting electrospinning solutions.Seamless and complex 3D shapes can be produced and by depositing fibrils of electrospun collagen along a defined axis, the alignment and distribution of cells within a bioengineered organ can be theoretically regulated to a high degree.This characteristic has direct implications in the fabrication and function of many different tissues, including musculoskeletal organs.By supplementing the electrospun collagen with other matrix constituents, growth factors and/or other peptides, the biological properties of a matrix can be exquisitely tailored to a specific tissue or bioengineering application.These characteristics provide enormous flexibility to the tissue Licensee OA Publishing London 2012.Creative Commons Attribution License (CC-BY) Competing interests: none declared.Conflict of interests: none declared.
All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.
All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.For citation purposes: Oryan A, Moshiri A, Sharifi P. Advances in injured tendon engineering with emphasis on the role of collagen implants.Hard Tissue.2012 Dec 29;1(2):12.
engineering process.As a derivative of a natural, highly conserved and non-immunogenic ECM protein, the electrospun collagen clearly has the potential to be used in the therapeutic delivery of stem cells, vascular bioengineering, hard and soft tissue reconstruction, wound care and drug delivery 57 .
Liao et al. 49 used combined electrospinning and mineralization procedure to process a series of collagen nanofibrous scaffolding systems with desirable characteristics suitable for biomimetic bone tissue engineering.They showed that the electrospun scaffolds had high surface area, high porosity and well-connected open pore network 49 .Bashur et al. 58 showed that the oriented micron-scale poly (d,l-lactico-co-glycolic acid) (PLGA) fibre meshes formed by the electrospinning process could regulate cell morphology.They suggested that electrospun fibre meshes may be suitable for the development of an engineered tendon and ligament tissue.The collagen functionalized thermoplastic polyurethane nanofibres (TPU/ collagen) were successfully produced by co-axial electrospinning technique with a goal to develop biomedical scaffold.Their in vitro results demonstrated that co-axial electrospun composite nanofibres had the characters of native ECM and may be used effectively as an alternative material for tissue engineering and functional biomaterials.The feasibility and efficacy of using core-shell composite nanofibres in improving cell-scaffold interactions has recently been demonstrated 15 .
Kim et al. 21developed a novel fabrication technique aiming to produce 3D macroporous and nanofibrous hyaluronic acid (HA) scaffold by an electrospinning process.They formed extensive fluffy nanofibre morphology for the first time that has been used to spontaneously generate a 3D nanofibrous structure.By simultaneous deposition of salt particulates as porogen during electrospinning and subsequent chemical cross-linking and salt leaching, a water-swellable, HA-based scaffold retaining macroporous and nanofibrous geometry could be produced.Bovine chondrocytes were cultured on the HA/collagen scaffold to assess the scaffold's cytocompatibility.The results revealed that cellular adhesion and proliferation were enhanced in proportion to the content of collagen, and the seeded chondrocytes maintained the roundness characteristic of a chondroblastic morphology 21 .
Telemeco et al. 57 characterized infiltration of the interstitial cells into tissue engineering scaffolds prepared with electrospun collagen using the natural polymer type I collagen from calf skin, electrospun type I gelatin, electrospun PGA, electrospun polylactic acid (PLA) and an electrospun PGA/ PLA co-polymer.Each of these materials was then electrospun into a cylindrical construct with a 2-mm internal diameter with a wall thickness of 200-250 ml.Electrospun scaffolds of collagen were rapidly, and densely, infiltrated by the interstitial and endothelial cells when implanted into the interstitial space of the vastus lateralis muscle of rats.Functional blood vessels were evident within 7 days.In contrast, implants composed of electrospun gelatin or the bioresorbable synthetic polymers were not infiltrated to any great extent and induced fibrosis.They showed that the topographical features, unique to the electrospun collagen fibril, promote cell migration and capillary formation.

Cross-linking and stabilization of the collagen scaffolds
One of the major deficiencies of the tissue-engineered collagen scaffolds are inferior biomechanical properties, making their usage difficult in clinical and experimental studies.Recently, some investigators have focused on the biomechanical properties of these scaffolds and improved them so that the scaffold with these characteristics could be used for ligament and tendon engineering technologies 38,51,59 .Brodie et al. 10 used adhesive-coated biological scaffolds such as bovine pericardium or PD tissues to augment primary suture repair of transected Achilles tendons aiming to improve the biomechanical performance of the graft.These adhesive constructs were wrapped around the transected cadaveric PD tissue.Their clinical results showed that these constructs were able to secure the biological scaffolds to the transected porcine tendons while improving their biomechanical properties when compared with the primary suture repair alone 10 .
Cross-linking and the resultant changes in the mechanical properties have been shown to influence the cellular activity within collagen biomaterials.Many techniques have been used to produce cross-linkage in tissue-engineered products, aiming to improve the biomechanical ability and stability of the artificial tissues.Most of these techniques were successful in producing cross-linking in the scaffolds; however, some of them, especially the chemical techniques, have some adverse effects on viable tissues.
To improve the mechanical properties and degradation rates of collagen scaffolds, chemical cross-linking is commonly employed.Stability and strength of the collagen scaffolds used for tissue engineering must satisfy their intended biomedical needs.Methods for stabilizing collagen-based materials with catechol-containing monomers have been developed to produce fibres with mechanical properties in tension comparable to those of the normal tendon.All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.
All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.For citation purposes: Oryan A, Moshiri A, Sharifi P. Advances in injured tendon engineering with emphasis on the role of collagen implants.Hard Tissue.2012 Dec 29;1(2):12.
biologically relevant mono-catechols indicated that the bi-catechol functionality of NDGA was responsible for the generation of superior tensile properties.Elimination of the nonreactive intermediates from the treated fibres with ethanol increased the effectiveness of the cross-linking process while simultaneously sterilizing the material.Catalysing oxidation by saturating the reaction buffer with oxygen increased the effectiveness of polymerization and the resulting tensile properties of the treated fibres.The material properties of the NDGA cross-linked fibres exceed the properties of collagen fibres treated with other cross-linking strategies such as glutaraldehyde and carbodiimide.These results indicated that NDGA crosslinking may provide a viable approach in stabilizing the collagenous materials for use in repair of ruptured, lacerated or surgically transected tendons 60 .
Cross-linking of the CS with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) provides a means of easily tailoring a scaffold's biostability and mechanical properties.Yunoki et al. 61 developed the thermal stability of a gel prepared from salmon atelocollagen aiming to improve crosslinking of the gel fibres, using EDC.Their results suggested that the gel employed for biomaterials can be fabricated from fish collagen by EDC crosslinking during fibril formation 61 .Powell and Boyce 62 developed the methods of CS cross-linking.They cross-linked the freeze-dried collagen-GAG (CG) sponges with increasing concentrations of EDC.Intermediate concentrations of EDC increased CS stability and strength while providing an environment in which cultured cells can attach, proliferate and organize in a manner conducive to dermal and epidermal regeneration 62 .Haugh et al. 63 investigated the effects of cross-linking on both the compressive modulus of CG scaffolds and the activity of osteoblasts seeded within them.EDC and glutaraldehyde cross-linking treatments were first investigated for their effect on the compressive modulus of the scaffolds.EDC and glutaraldehyde treatments produced the stiffest scaffolds (four fold increase when compared with dehydrothermal cross-linking).When cells were seeded onto the scaffolds, the stiffest scaffolds also showed increased cell number and enhanced cellular distribution when compared with the other groups.Taken together, these results indicated that crosslinking can be used to produce CG scaffolds with a range of compressive moduli, and that increased stiffness enhances cellular activity within the scaffolds 63 .The bio logical scaffolds used in tissue engineering are incorporated in vivo by a process of cellular in-growth, followed by host-mediated degradation and replacement of these scaffolds, in which phagocytic cells from the monocyte/macrophage cell lineage play a key role.Chemical degradation of the scaffolds with collagenases is well established, but to date this has not been correlated with an in vitro model of cell mediated scaffold degradation.A murine monocyte/macrophage cell line was cultured on collagen scaffolds cross-linked by dehydrothermal treatment (DHT) or carbodiimide (EDC).These cells attached to collagen scaffolds proliferated and exhibited macrophage aggregation to form giant cells.Crosslinking of the scaffolds by DHT or EDC increased the resistance of the scaffolds to degradation by macrophages.Increasing the amount of cross-linking in the scaffolds made them more resistant to degradation by collagenase 64 .
Physical cross-linking is another technique to improve the biomechanical performance of the engineered tissues although it seems that the cross-linked tissues from these techniques have inferior biomechanical performance.Some of the investigators developed these techniques for skin tissue engineering technologies.Torres et al. 65 showed that the distribution of cells and non-uniformity of contraction in the CG sponges vary with cross-linking treatment.Their results indicated that physical crosslinking methods such as UV irradiation can be effectively used to control the elastic modulus and as a result, the cell-mediated contraction of the sponges 65 .
Kanungo and Gibson 59 used a vacuum filtration technique to increase the volume fraction of solids in the slurry.Their result showed that this technique increased the density and mechanical properties of the CG scaffolds with some characteristics appropriate for soft tissue growth.They showed that attachment of the cells to the scaffold was directly proportional to the specific surface area of the scaffold, which defines the total internal surface area per volume of a scaffold.

Hybridization of the collagen-based bioimplants
Many biomaterials are used to improve tendon healing alone or in combination with collagen matrices to increase the efficacy of the engineered collagenbased scaffolds in tissue regenerative medicine with some significant promising curative effects in vitro and in vivo.The most influent biomolecules and biostructures discussed here are the broad range of growth factors, GAGs and stem cells.

Growth factors
Growth factors are signalling molecules that are involved in control of cell growth and differentiation.The most important growth factors in tendon healing are basic fibroblast growth factor (bFGF or FGF), vascular endothelial growth factor (VEGF) and insulinlike growth factor 1,7-9 .A complex system exists, whereby growth factors may have multiple dose-dependent effects and act synergistically with other growth factors.The importance of growth factors as regulators of phases of tendon healing has been well established 1,[7][8][9] .Wounding and inflammation provoke release of growth factors from platelets, polymorphonuclear leukocytes and macrophages 9 .

Licensee OA Publishing London 2012. Creative Commons Attribution License (CC-BY)
Competing interests: none declared.Conflict of interests: none declared.
All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.
All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.For citation purposes: Oryan A, Moshiri A, Sharifi P. Advances in injured tendon engineering with emphasis on the role of collagen implants.Hard Tissue.2012 Dec 29;1(2):12.
Growth factors bind to cell surface receptors resulting in intracellular changes in DNA synthesis and expression.The net result is induction of neovascularization and chemoattraction, along with stimulation of fibroblast proliferation and collagen synthesis 7,8 .These changes in turn influence the healing cascade.
Much research has focused on developing VEGF delivery systems to enhance angiogenesis in wound repair and in tissue engineering.Collagen can be used as a delivery system because of its biocompatibility, but its fast degradation rate and limited affinity with growth factors are disadvantageous for maintaining a sufficient growth factor concentration in injury sites.Therefore, this could be determined as a new area of research in tendon tissue engineering technologies.
An important issue in tissue engineering is vascularization of the implanted construct, which often takes several weeks.In vivo, the growth factors VEGF and FGF2 show a combined effect on both angiogenesis and maturation of blood vessels 1,7,8 .It has been shown that addition of these growth factors to an acellular construct increases blood vessel formation and maturation.Nillesen et al. 23 prepared and characterized five collagen-based porous scaffolds with different combinations of heparin, bFGF and VEGF.The scaffolds were then subcutaneously implanted in 3-month-old Wistar rats.Of all the scaffolds tested, the one with a combination of growth factors displayed the highest density of blood vessels (type IV collagen) and more mature blood vessels (smooth muscle actin).In addition, no hypoxic cells were found in this scaffold at day 7 and day 21 post-operation.It has been indicated that addition of both FGF2 and VEGF to an acellular construct enhances an early mature vasculature 23 .
To enhance VEGF binding to collagen scaffolds and reduce the collagen degradation rate, a simple way to modify porous collagen-based scaffolds is the chemical addition of sulfhydryl groups, which then allow both crosslinking of the collagen fibres with each other and immobilization of more VEGF in the scaffold after treatment with sulfo-SMCC has been introduced. 66t has been demonstrated that crosslinking led to a slower degradation rate of the collagen scaffolds, whereas cellularization was improved by both cross-linking and presence of the VEGF.Angiogenesis was increased only moderately by cross-linking, but significantly more by the presence of the immobilized VEGF.The authors showed that chemical conjugation of collagen scaffolds to VEGF by Traut's reagent and sulfo-SMCC is an effective delivery system in wound repair and tissue engineering and the biological activity of VEGF was not obviously reduced by cross-linking 66 .
Nillesen et al. 24 developed a method to design an acellular double-layered skin construct, using matrix molecules and growth factors to target specific biological processes.The epidermal layer was prepared using type I collagen, heparin and FGF, whereas the porous dermal layer was prepared using type I collagen, soluble elastin, dermatan sulphate, heparin, FGF and VEGF.The construct was biochemically and morphologically characterized and evaluated in vivo using a rat full-thickness wound model.The double-layered skin construct showed more cell influx, significantly less contraction and increased blood vessel formation at early time points in comparison with the commercial scaffold (Integra DRT) and/or the untreated wound.On day 14, the double-layered skin construct also had the fewest myofibroblasts present.On day 112, the double-layered skin construct contained more elastic fibres than Integra DRT and the untreated wound.Structures resembling hair follicles and sebaceous glands were found in the double-layered skin construct and the untreated wound, but hardly any were found in Integra DRT 24 .
Shi et al. 67 activated CM with engineered human bFGF and implanted it to repair a defect in the abdominal wall in rats.It was shown that bFGF effectively accelerated reconstruction of the injured abdominal wall with minimal adhesion, and the mechanical strength of the implants was also improved.

CG scaffolds
Classically, the cells used in tissue engineering are seeded onto the collagen gel scaffolds.Contraction of the gel is followed by alignment and reorganization of the matrix.Less attention has been paid to characterizing the scaffold microstructure and mechanical properties in comparison with the processing and bioactivity of scaffolds.Despite substantial improve ment in the cross-linking and stabilization methods, such constructs are never able to fully reproduce the complex architecture of tendon and as such remain biomechanically inferior.Recently, attention has focused on the role of proteoglycans and GAGs in tendon structure and function.Incorporation of these components into scaffolds may be required to reproduce the architecture more closely to that of the normal tendon 3,7,9 .GAGs are bioactive biomolecules that have a strong capacity for combining with collagen molecule in developing tissue engineered scaffolds and composites 68,69 .It has been shown that the effect of osteoblasts on CG scaffolds is affected by collagen and GAG concentrations 69 .The cellular structure of the CG scaffolds, used in tissue engineering, must be designed to meet the biocompatibility, degradability, pore size, pore structure and specific surface area 46,68 .Having several biocharacteristics in GAGs make them valuable molecules for incorporation in collagenous biomaterials.To prepare tailormade CG matrices with a well-defined biodegradability and bioavailable GAG content, the cross-linking conditions have to be controlled.Additionally, the ultrastructural location of GAGs in the engineered substrates should resemble that of the application site 70 .

Licensee OA Publishing London 2012. Creative Commons Attribution License (CC-BY)
Competing interests: none declared.Conflict of interests: none declared.
All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.
All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.For citation purposes: Oryan A, Moshiri A, Sharifi P. Advances in injured tendon engineering with emphasis on the role of collagen implants.Hard Tissue.2012 Dec 29;1(2):12.
A well-characterized CG matrix functions as an ECM analogue of musculoskeletal tissues 13 .
Pieper et al. 71 developed a techniques to produce CG scaffolds.They used chondroitin sulphate as a model of GAGs.In their technique, CS was covalently attached to collagen using EDC and N-hydroxysuccinimide (NHS).General applicability of EDC/NHS for immobilizing GAGs was demonstrated with dermatan sulphate, heparin and heparan sulphate.These matrices revealed comparable physico-chemical characteristics, biodegradabilities and preserved bioavailable GAG moieties.At the ultrastructural level, they were observed throughout the matrix fibres and at the outer sites, and located, either parallel or orthogonally, at the periphery of the individual collagen fibrils.They explained that the compositional and ultrastructural similarity between the matrices and tissue structures like tendon, cartilage and basement membranes can be realized after attachment of specific GAG types 71 .
The beneficial effects of acid hyaluronic or sodium hyaluronate (HA) as another GAG molecule on tendon healing has been well established 1,72 .It is one of the best biomaterial options to hybridize collagen-based bioimplants.It has been used to develop the efficacy of collagen-based tissue engineered products in clinical applications.In an in vitro study, hyaluronan has been shown to promote adhesions between cells and the substrate and stabilize these adhesions 73 .Hyaluronidase-treated cells adhered to the substrate less efficiently, forming fewer adhesion sites, which, once formed, grow rapidly to large sizes at the cell periphery 73 .In addition, the effects of HA on cell proliferation and chondroitin sulphate synthesis by chondrocytes embedded in collagen gels have been shown previously 74 .Similar effects have been shown in tenocytes in vivo 72 .
Fibre-based scaffolds have been widely used for tendon and ligament engineering.The knitted scaffolds have been proved to favour collagenous matrix deposition which is crucial for tendon and ligament reconstruction.However, such scaffolds have the limitation of being dependent on a gel system for cell seeding, which is unstable in a dynamic environment such as the knee joint.Different types of hybrid scaffolds, based on the knitted biodegradable polyester scaffolds, aiming to improve the mechanical properties and cell attachment and proliferation on the scaffolds have been developed by Sahoo et al. 75 It has been demonstrated that coating techniques could modulate the mechanical properties and facilitate cell attachment and proliferation in the hybrid scaffold, which can be applied in tissue engineering of tendon and ligaments.They have also shown that collagen is particularly effective in aiding cell attachment, growth and proliferation 75 .
Collagen-elastin scaffolds may be valuable biomaterials for tissue engineering because they combine tensile strength with elasticity.Collagen and elastin networks contribute to highly specialized biomechanical responses in numerous tissues and species 76 .By incorporating reinforcing collagen microfibres, the recombinant elastomeric protein-based biomaterials can play a significant role in the loadbearing capacity of the substituted tissue.It has been indicated that similar composites can be incorporated into tissue engineering schemes that seek to integrate cells within the structure, prior to or after implantation in vivo 36 .Caves et al. 36 described a process for fabrication of the vascular grafts from a recombinant elastin-like protein reinforced with collagen microfibres that facilitated control over collagen microfiber orientation and density.In turn, fibre architecture and processing of the elastin-like protein modulated the suture retention strength, burst strength and compliance.Iterative adjustments to the fibre layout led to a design that met mechanical targets.The fabrication scheme employs a non-thrombogenic elastin-like protein with the possibility that these structures may be advantageous as vascular graft implants without further modification.In addition, modification of the scheme to incorporate living cells ex vivo or in situ may be feasible 36 .

Stem cells and collagen-based scaffolds
Tendon defects remain a major concern in plastic surgery because of the limited availability of tendon autografts.Although immune rejection prohibits use of tendon allografts, replacement of most of the prosthetics fails to achieve satisfactory longterm results in tendon repair 77 .The tissue engineering technique, however, can generate different tissues using autologous cells and thus may provide an optimal approach to address this concern.Tissue engineering of skeletal tissues from cultured cells has been attempted using a variety of synthetic and natural macromolecular scaffolds 78 .Collagen has been found to be the best biomaterial option for cell seeding 79 .Kroehne et al. 78 applied artificial CS consisting of collagen I with parallel pores using permanent myogenic cell line C2C12 and showed that the biodegradable CS with parallel pores supported maximum formation of the oriented muscle fibres and were highly compatible with force generation in the regenerated muscle 78 .
Recently, stem cell-based therapy has become more popular among investigators and surgeons in the area of tendon healing and to date, many studies have used stem cells in their tissue engineering technologies, aiming to improve the healing process of lesions.However, the recruitment of native autologous stem cells at the targeting site has not been sufficient, which limits clinical application of autologous stem cells.Biomaterials have been increasingly used in tissue repair.The feature of the ideal scaffolds to improve cell seeding technologies such as porosity, cross-linking and fibre alignment have been Licensee OA Publishing London 2012.Creative Commons Attribution License (CC-BY) Competing interests: none declared.Conflict of interests: none declared.
All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.
All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.For citation purposes: Oryan A, Moshiri A, Sharifi P. Advances in injured tendon engineering with emphasis on the role of collagen implants.Hard Tissue.2012 Dec 29;1(2):12.discussed earlier in this review.Moreover, there are several other techniques that improved cell seeding technologies.Shi et al. 80 covalently conjugated the collagen scaffold with anti-cardiomyocyte stem cell antibody and could capture and enrich the positive stem cells both in vitro and in vivo.After implantation in the injured myocardium, regeneration of the cardiomyocytes was effectively enhanced.This work conducted an exploratory investigation for enriching native autologous stem cells to biomaterials at the wound site 80 .
Biomaterials not only serve as scaffolds for cell proliferation and differentiation but also provide guidance for 3D re-establishment.The stem cells could be broken down into three different types by their potencypluripotent, multi-potent and less potent stem cells.The pluripotent stem cells such as the embryonic stem cells have established a new area of research.These cells are highly mitotic and could be helpful in tissue regeneration, although till date, there are limited technologies aiming to control their behaviour of differentiation.Application of these cells in the tendon defects may not result in tendon-like tissue, and several possibilities exist to initiate their differentiation into other tissues.In addition, by application of this type of stem cells, the repair process could be influenced by the host immune rejection.Therefore, application of these cells in the potent and less potent stem cells such as autologous mesenchymal stem cells (MSCs) are now used in tissue regenerative medicine, especially in tendon engineering technologies aiming to produce a tendon-like tissue in vitro and improve tendon healing in vivo with some curative effects [17][18][19]35,[81][82][83] . As theirpotency decreases, these two later types of stem cells differentiate further, providing additional possibilities of application in tissue regenerative medicine aiming to produce a tendonlike tissue with much fewer side effects.
Successful tissue-engineered repair in ageing adults requires an abundant source of autologous, multi-potent MSCs.The number of bone marrowderived MSCs declines dramatically with ageing, but their efficacy would not decrease with ageing.Dressler et al. 84 showed that MSCs did not lose their benefit as a tendon repair therapy with ageing in rabbit and showed that the MSCs could be cryogenically stored for 3 years and still effectively repair soft tissue injuries.So, these cell types are frequently used in tissue engineering technologies 79 .
Butler et al. 13 developed the seeded CG matrix technique, using autologous keratinocytes cultured to subconfluence and concluded that a cell population enriched with proliferating cells can be expected to generate an epidermis more efficient than the uncultured epithelial cells when seeded into a CG matrix graft.Cultivation of keratinocytes also made massive epithelial expansion possible.Thus, combining the seeded CG matrix technique with the cellular expansion of in vitro cultivation would allow for reconstruction of large surfaces from small skin donor sites 13 .
Gentleman et al. 35 showed that a combination of stem cells and collagen scaffold/collagen gel composites could be designed and determined the key properties of native ligament/tendon tissue.They demonstrated that the presence of cells significantly enhanced the biomechanical properties of collagen scaffolds in vitro.
Awad et al. 81 isolated MSCs from the bone marrow of adult New Zealand white rabbits.The cells were then culture expanded, suspended in type I collagen gel and implanted in a surgically induced defect in the donor's right patellar tendon.A cell-free collagen gel was then implanted into an identical control defect in the left patellar tendon.They showed that delivering a large number of MSCs in the wound site can significantly improve its biomechanical properties by only 4 weeks but produce no visible improvement in its microstructure.Their results indicated that implants prepared at higher seeding densities showed more well aligned and elongated cell nuclei after 72 h of contraction.They concluded that changes in the nuclear morphology of the MSCs in response to the physical constraints provided by the contracted collagen fibrils may then trigger differentiation pathways towards the fibroblastic lineage and influence cell synthetic activity and biomechanical performance of the graft.They suggested that controlling the contraction and organization of the cells and matrix is critical for successfully creating tissue-engineered grafts 17 .
In another study, collagen gels were seeded with rabbit bone marrowderived MSCs and contracted onto sutures at initial cell densities of 1, 4 and 8 million cells/ml 18 .These MSC-collagen composites were then implanted in similar defect models of patellar tendons of the animals providing the cells.These surgically implanted, tissue-engineered MSCcollagen composites significantly improved the biomechanical properties of the repair area of the tendon, although greater MSC concentrations produced no additional significant histological or biomechanical improvement.In a similar study, Juncosa-Melvin et al. 19 fabricated autogenous tissue-engineered constructs at four cell-to-collagen ratios by seeding MSCs from adult rabbits.They implanted the constructs into bilateral full-thickness, full-length defects created in the central third of the patellar tendon.Twenty weeks after surgery, their results indicated that the repair area achieved higher maximum forces than in the previous studies and without ectopic bone, but they still needed to achieve sufficient stiffness 19 .They continued their study on patellar tendon defect in the rabbit and showed that mechanical stimulation of stem cell-CS constructs can significantly improve the biomechanical properties of the injury site of the tendon up to and well Licensee OA Publishing London 2012.Creative Commons Attribution License (CC-BY) Competing interests: none declared.Conflict of interests: none declared.
All authors contributed to the conception, design, and preparation of the manuscript, as well as read and approved the final manuscript.
All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.For citation purposes: Oryan A, Moshiri A, Sharifi P. Advances in injured tendon engineering with emphasis on the role of collagen implants.Hard Tissue.2012 Dec 29;1(2):12.
beyond the functional limits of in vivo loading 83 .
Although bone marrow and lipid MSCs have frequently been used, the autologous tenocytes have been shown to be more effective in tendon engineering than other cell sources.The feasibility of engineering the tendon tissues with autologous tenocytes to bridge a tendon defect in either a tendon sheath open model or a partial open model in a hen model has been demonstrated by Cao et al. 77 They harvested flexor tendons from the left feet of 40 hen and digested them with 0.25% type II collagenase.The isolated tenocytes were expanded in vitro and mixed with unwoven poly-glycolic acid fibres to form a cell-scaffold construct in the shape of a tendon.The constructs were wrapped with intestinal sub-mucosa and then cultured in Dulbecco's modified Eagle medium plus 10% foetal bovine serum for 1 week before in vivo transplantation.A defect of 3-4 cm was created in the feet at the second flexor digitorum profundus tendon by resecting a tendon fragment.The defects were bridged with a cell-scaffold construct in the experimental group.The specimens were harvested at 8, 12 and 14 weeks post-injury.At 14 weeks, the engineered tendons displayed a typical tendon structure hardly distinguishable from that of the normal tendons.Biomechanical analysis demonstrated increased breaking strength of the engineered tendons with time, which reached 83% of normal tendon strength at 14 weeks 77 .
Human embryonic stem cells (hESC) and their differentiated progenies are another attractive cell source for transplantation therapy and tissue engineering.The efficacy of MSCs derived from hESC within a knitted silk-CS scaffold in promoting tendon regeneration has been investigated by Chen et al. 85 When subjected to mechanical stimulation in vitro, these cells exhibited tenocyte-like morphology and positively expressed tendon-related gene markers as well as other mechano-sensory structures and molecules.In ectopic transplantation, the tissue-engineered tendon under in vivo mechanical stimulus displayed more regularly aligned cells and larger collagen fibres.This in turn resulted in enhanced tendon regeneration in situ, as evidenced by improved histological scores and superior mechanical characteristics.Cell labelling and ECM expression assays demonstrated that the transplanted embryonic stem cells not only contributed directly in tendon regeneration but also exerted an environment-modifying effect on the implantation site in situ 85 .

Discussion
The authors have referenced some of their own studies in this review.These referenced studies have been conducted in accordance with the Declaration 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.All human subjects, in these referenced studies, gave informed consent to participate in these studies.Animal care was in accordance with the institution guidelines.Tissue-engineered tendon implants have the potential to significantly improve the healing potential of the tendon and ligament injuries, especially those associated with tumours, trauma, and congenital or acquired deficiencies where autograft or allograft tissues might not be available in sufficient quantity and quality for reconstruction.
This review has strongly shown that collagen-based scaffolds/implants could be selected as the material of choice in tendon or ligament tissue engineering technologies to produce tendon-like grafts.The reason for this statement could be related to the potency of the collagen molecules to provide the ideal criteria, explained earlier in this review.From the findings of previous investigations, it can be concluded that tissue engineering technologies have come a long way and the revolution in the area of transplantation can be expected in the near future.This study has demonstrated that the potency of collagen scaffolds/ implants could be improved by designing new strategies regarding tendon engineering technologies.Applications of combined bioimplants with biological and biotechnologic molecules and different types of stem cells with collagen-based scaffolds possibly could be desirable in reconstructing the structural and physical properties of the tendon.
Fabrication of these bioimplants could be suggested as a new area of research to produce and supply a scaffold that is structurally and functionally comparable to normal tendons for clinical application.To access this step, first of all, the blind points present in previous researches should be clarified.Future studies should focus on the in vivo curative effects of these tissue-engineered scaffolds and bioproducts by performing experimental tendon defect models.Unfortunately, most research in this area has been performed in vitro and because of reasons such as host immune reaction and toxicity, these results are not sufficient to approve application of these tissue-engineered products as a safe product and with promising curative effects in vivo and in clinical trials.Most evaluation techniques and methodologies in this field could not explain the healing processes and focused on the tissue-engineered products and not the defect area.With regard to previous studies by Oryan and Moshiri, 7 Moshiri and Oryan 8 and Oryan et al. 72 , it could be strongly suggested that the investigators should shift from some microanalytical evaluations such as light microscopy to ultrastructural analysis such as transmission and scanning electron microscopic studies 7,8,72 .
Most of the researches in the field of collagen-based tissue engineering technologies have spent their efforts to fabricate novel bioimplants possessing the following characteristics:• They should be highly biocompatible and have rapid-moderate absorption rate.• They should be acellularized to generate a minimum host cell immune reaction.• They should have high homogenous porosity and high affinity to bind with seeded cells and allow them to move in their architecture.• They should be highly aligned and well cross-linked collagen fibres to perform a strong biomechanical performance.
studied the biocompatibility, tissue response and collagen fibre orientation of the collagen Licensee OA Publishing London 2012.Creative Commons Attribution License (CC-BY) Competing interests: none declared.Conflict of interests: none declared.
studied the influence of fibre alignment and scaffold architecture on cellular interactions and matrix organization.They fabricated three scaffolds from the photo-cross-linkable elastomer poly-glycerol-sebacate, with changes in fibre alignment of nonaligned versus aligned scaffolds.The neonatal cardiomyocytes, used as a representative cell type, that were seeded onto the scaffolds maintained their viability and aligned along the surface of the fibres.Briefly, their Licensee OA Publishing London 2012.Creative Commons Attribution License (CC-BY) Competing interests: none declared.Conflict of interests: none declared.
Fibres produced from pepsin-solubilized bovine tendon type I collagen were polymerized with the di-catechol nordihydroguaiaretic acid (NDGA) by Koob and Hernandez 60 .NDGA resulted in a dose-dependent increase in the tensile strength and stiffness of type I collagen fibres.A second treatment with NDGA improved the tensile properties significantly.Comparison of the effects of NDGA with those of Licensee OA Publishing London 2012.Creative Commons Attribution License (CC-BY) Competing interests: none declared.Conflict of interests: none declared.