Tendons as a strategy for mechanical augmentation and repair

Introduction

Pure collagen, as a non-living materials from human or animal sources, is a biomaterial prominently employed in tissue engineering, regenerative drugs, dentistry, and pharmaceutical purposes. The formation of covalent crosslinks between structural moieties at varied hierarchical ranges in collagen induces further molecular stability resulting in augmentation of mechanical properties of the collagen-based supplies. As mentioned in some current opinions,^1–4 chemical or bodily crosslinking strategies have turn out to be a typical instrument for reinforcing mechanically the engineered collagen scaffolds, implants, or units and enhancing their resistance to enzymatic degradation.

The crosslinking of native collagen whereas situated within the physique is a very completely different subject, which purports two sorts of processes, endogenous and exogenous. The endogenous pure enzyme-driven crosslinking of dwelling collagen is prime to the maturation and therapeutic processes in our organism, and to the event and performance of connective tissues. Lysyl oxidase (LOX) is the enzyme accountable for this basic course of that’s mainly outdoors our management. The endogenous nonenzymatic crosslinking between adjoining collagen fibrils stabilizes the community and is believed to be mediated by macromolecules resembling proteoglycans and fibronectin. One other endogenous nonenzymatic crosslinking is because of growing old, and is pushed by a course of triggered by the superior glycation end-products (often known as AGEs). It induces brittleness, much less resistance to break, and impaired matrix remodelling of the connective tissues, and is due to this fact thought-about deleterious, regardless of resulting in enhanced stiffness of tissular collagen.

The exogenous crosslinking of native collagen has doubtless been impressed from the crosslinking of engineered collagen-based supplies, and is turning into a doubtlessly therapeutic technique for treating issues of the connective tissue. It implies the direct publicity at particular anatomic places of the dwelling collagenous tissues to appropriate chemical, photochemical, enzymatic, or thermal processes, leading to structural modifications of the collagen macromolecules, that are accompanied by the anticipated or supposed adjustments in properties. In different phrases, the crosslinks are launched on objective so as to modulate favorably the mechanical and physiological properties of the native tissue. Nonetheless, it’s important to establish and overcome the organic and technical challenges to the interpretation of crosslinking methodology right into a practicable scientific remedy.

So far, a profitable software of an exogenous crosslinking course of has been the photochemical crosslinking of the corneal collagen within the eye,^5–7 which is at present the routine remedy for keratoconus, an ophthalmic situation that may result in extreme visible incapacity if left untreated. By irradiating the cornea with ultraviolet (UV)-A rays (wavelengths 315–400 nm, photon energies ~3 to ~ 4 eV), within the presence of a photoinitiator, the development of keratoconus is arrested because of the stiffening of corneal collagen induced by a radiation-induced crosslinking response. We have now proposed to increase the tactic to the remedy of eyelid laxity (eg, floppy eyelid syndrome), and have proven^8,9 in ex-vivo animal (ovine) tissue that the UV-A-induced crosslinking of tarsal collagen led to elevated mechanical power and stiffening of the tissue. We additionally demonstrated that the mechanical properties of ex-vivo porcine aortic peripheral collagen (residing in tunica adventitia of the vessel) have been considerably enhanced following irradiation with UV-A rays, and proposed the method as a possible technique for stopping the rupture of stomach aortic aneurysms.¹⁰ In a subsequent examine,¹¹ the aortic specimens have been subjected to in-vitro collagenolysis after which subjected to mechanical analysis, with or with out irradiation. We discovered not solely that crosslinking could be useful to the degenerated wall, but additionally that the sooner the irradiation the extra immune to rupture would be the wall.

On this overview, we analyzed the literature printed on using exogenous collagen crosslinking in tendons. We expound on the power of crosslinking to arrest propagation of partial tears by augmenting mechanical properties, to impart stabilization to the tissue, and thus to contribute to the therapeutic of injured or lax tendons. The crosslinking applied sciences utilized for the fabrication of collagen-based tissue-engineered scaffolds and units to restore torn tendons or ligaments should not a subject of the current assessment.

Chemical Crosslinking of Tendons

Some many years in the past, Haut¹² reported that the quantitative discount of pure crosslinks within the collagen of experimental animals subjected to a lathyritic food regimen was related to the lower of mechanical power and elastic modulus of the rat-tail tendons. Dietary lathyrogens (eg, β-aminopropionitrile) are recognized to inhibit the exercise of LOX,^13,14 resembling impairing the covalent crosslinking, however not the biosynthesis of collagen, within the physique. However this related discovering, the thought to crosslink the tendinous tissue so as to make it stronger took one other twenty years to emerge.

The primary examine trying to determine the significance of exogenous collagen crosslinking associated to tendons was printed by Zhao et al¹⁵ on the Mayo Clinic in Rochester, MN. Contemplating the issues related to the suture–tendon interface (recurrent tissue rupture, suture rupture, or suture failure resulting from low pullout resistance), the investigators prompt the injection of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, see construction in Determine 1) as a crosslinking agent into the interface of 24 ex-vivo canine flexor digitorum tendons. A tendon specimen that was pliable grew to become a inflexible rod after 1 h in 10% aqueous EDC. Mechanical analysis confirmed a rise in final power however no important change in stiffness. All samples failed the suture pullout exams, which didn’t hamper the investigators’ optimism. In experiments reported later by the identical group,¹⁶ the sutures have been impregnated with EDC, such avoiding injection however utilizing the identical tendon mannequin. Nonetheless, the outcomes have been just like these reported beforehand.¹⁵ All specimens failed the suture chopping exams. The evaluation of cell viability indicated a good impact on the numbers of viable cells (cultured tenocytes). It’s not clear whether or not EDC was first adsorbed onto the suture and after that launched into the tendon’s interfacial area. Because the chemical nature of the sutures was not disclosed, it’s troublesome to establish which of the physisorption or chemisorption processes ruled formation of a coating.

Determine 1 Chemical buildings of brokers generally used for crosslinking the collagenous tissues.

One other pioneering work was carried out by Magnusson’s group,17 then on the Institute of Sports activities Medication in Copenhagen, who investigated the mechanical results induced by the crosslinking of rat-tail tendon with glutaraldehyde (GA, Determine 1) at two structural ranges of the tendon hierarchy, the fascicles and the fibrils. The response was performed at 4°C in diluted aqueous medium for twenty-four h. Mechanical analysis was carried out utilizing atomic pressure microscopy (AFM) in three completely different procedures for tendon fibrils, and uniaxial tensile check for fascicles. In fibrils, the crosslinking induced a considerable enhance of mechanical traits, particularly of Younger’s modulus (YM), an oblique measure of stiffness. Vital will increase in YM, yield stress, and yield pressure have been additionally measured for the tendon fascicles. The authors concluded that crosslinking is vital in offering further power to tendons. Additionally they famous that fibril power induced by crosslinking seems to surpass that induced in fascicles, which is vital contemplating that the fibril is the first load-bearing unit of a tendon, though the fascicles are hierarchically the most important.

To our data, the subsequent related physique of labor on tendon crosslinking has been carried out at ETH Zürich, in Snedeker’s Orthopedic Biomechanics Laboratory.^18–23 The investigators’ intention was to modulate in situ collagen crosslinks so as to enhance tendon therapeutic by arresting development of tearing. Extra exactly, they envisaged that the mechanical enhancement of tendons by exogenous collagen crosslinking might have such outcomes as:¹⁸ (a) an enchancment of suture retention; (b) reinforcing shoulder joint stability by stabilization of capsular ligaments; (c) to arrest development of partial rotator cuff tendon tears; or (d) to strengthen the marginal areas of partial tears, such restoring a good pressure distribution and rendering smaller tears manageable surgically. Of their first experiments, genipin (GP, Determine 1) was used as a crosslinking agent, which is a non-toxic pure substance remoted from crops of genera Genipa and Gardenia. Samples of each rat-tail tendon and equine digital flexor tendon that have been handled with GP confirmed a rise of their YM (stiffness), maximal tensile stress, and toughness. Genipin remedy was additionally utilized in an in vitro mannequin (equine tendon) of partial tendon tear/damage, when specimens of intact tendon, partially torn tendon, and GP-crosslinked tendon (3-day incubation) have been in contrast. The crosslinked tendon recovered a lot of the mechanical integrity misplaced within the torn specimens. In a subsequent examine,¹⁹ the consequences of two completely different chemical crosslinkers, GP and methylglyoxal (MG, Determine 1), have been in contrast in rat-tail tendon fascicles (RTTF) and equine superficial digital flexor tendon (ESDFT) specimens by analysis in a uniaxial mechanical tester. Beside direct measurements, two in-vitro fashions have been additionally employed for analysis (ESDFT load-bearing mannequin, and tear propagation below cyclic loading to failure mannequin). It was discovered that GP enhanced the power and stiffness in each tendons, whereas MG detrimentally affected the ESDFT, which was tentatively attributed to a sure hierarchical order of crosslink formation. Another parameters (toughness, yield pressure, hysteresis, and cyclic rest) displayed conflicting values. Within the investigators’ conclusion, GP confirmed essentially the most potential for arresting tear development and enhancing mechanical efficiency of handled tendons.

To additional promote GP as an acceptable chemical agent for exogenous crosslinking of the tendinous tissue, the ETH group carried out in depth quantitative analysis of putative related cytotoxicity.²⁰ Primarily based on cell viability, metabolic exercise and motility assays, gene expression evaluation, differential scanning calorimetry, and uniaxial mechanical testing, they’ve concluded that the mechanical augmentation by way of GP-induced crosslinking might be applied on the compromise of permitting some stage of cytotoxicity, a scenario that may be improved post-crosslinking by cell survival strategies. GP was additional evaluated within the contemporary ovine shoulder infraspinatus tendon, a longtime ex-vivo mannequin for rotator cuff tendon tears.²¹ The target of the examine was to see whether or not crosslinking can enhance the suture pullout power within the case of both easy single-loop sutures or modified Mason-Allen suture bridge approach sew patterns. A complete of 32 tendons have been used and 142 suture pullout exams have been carried out for each suture strategies. Two units of tendons have been incubated in a buffer containing 20 mmol/L GP at 36°C, for 4 h or 24 h, and following the remedy all specimens (together with untreated controls) have been evaluated uniaxially in a mechanical tester. For single-loop sutures, solely the 24-h remedy with GP led to a rise of the utmost pullout pressure (73 N vs 56 N), whereas the shorter remedy didn’t have any impact. For the Mason-Allen sew patterns, crosslinking didn’t present any profit. It was concluded that, within the case of single-loop sutures, the exogenous crosslinking with GP presents scientific relevance on the suture–tendon interface by enhancing the resistance to pullout, which can assist with the restore of rotator cuff tears. Publication of this examine attracted each curiosity and a few criticism.²² Thus, the examine didn’t focus on why the stitching configuration has an impact on the crosslinking final result. It neither offered a sign on the scientific utility, security and efficacy, or stage of therapeutic. Maybe essentially the most helpful suggestion²² was to implement a supply system for GP on the crosslinking website.

The relevance of suture–tendon interface was additional investigated in a newer examine from the ETH group.²³ Twenty-five ex-vivo bovine superficial digital flexor tendons have been processed into the next units: (a) wholesome tendons stitched with polyethylene sutures (single loop); (b) wholesome tendons stitched with GP-coated sutures; (c) specimens degraded by in-vitro collagenolysis and stitched with regular sutures; and (d) degraded tendons stitched with GP-coated sutures. Mechanical analysis was carried out 24 h after suturing. The coating with GP improved with statistical significance the suture pullout parameters, each pressure to failure (N) and work to failure (mJ), within the wholesome tendons, whereas in these degraded it was solely the pressure to failure that improved. Clearly the presence of genipin within the coating enhanced regionally the crosslinking of surrounding tendinous tissue the place the sutures have been confined. As acknowledged by ETH investigators, the coating used for sutures on this examine was really developed²⁴ by Hedman et al on the College of Kentucky, as a sustained launch system for the crosslinking agent. It consists of a fancy combination of GP, poly(lactic-co-glycolic acid), and poly(ethylene glycol) dissolved in acetone and dimethyl sulfoxide. It was proven that the ensuing layer is a diffusion kind system for the managed launch of an lively agent (GP) from a biodegradable polymer matrix, and this was checked each in vitro and ex vivo (equine tendons).²⁴ The tendons coming involved with the GP that resides on, or is launched from the sutures, displayed enhanced final tensile stress, load to failure, and stiffness. In a later examine,²⁵ the group investigated the intratendinous injection of GP in ex-vivo bovine superficial digital flexor tendons, both wholesome or degenerated (by in-vitro collagenolysis). The injectable resolution consisted of 80 mmol/L GP in an answer of buffer and dimethyl sulfoxide. The specimens have been cyclically loaded for 500 cycles after which loaded to failure. Whereas no important good thing about injection was seen within the wholesome tendons, the degraded specimens confirmed important will increase in final pressure, final stress, elastic modulus, work to failure, and pressure to failure. It was concluded {that a} degenerated tendon might be mechanically recovered by injection with GP and the process was prompt to deal with tendinopathies. Of their most up-to-date work,²⁶ the ETH group investigated the good thing about GP-coated sutures in human tendons. Human biceps lengthy head tendons have been harvested at operation from 25 sufferers. Polyethylene sutures, both regular or GP-coated, have been inserted in two units of tendons. Mechanical testing confirmed that the coated sutures have been related to greater forces to failure, however the different properties weren’t affected. Cell viability was a perform of the gap from the suture, with cytotoxicity manifested as much as about 3 mm to suture, and diminishing farther to normality.

Advantages of crosslinking tendons with GP have been additionally contemplated for a unique purpose. Maher’s group on the Hospital for Particular Surgical procedure in New York have investigated the protecting results in opposition to harm to tendon grafts attributable to gamma-ray sterilization.²⁷ The usage of donor tendon allografts to restore the rupture of anterior cruciate ligament requires their publicity to excessive doses of gamma radiation leading to harm to the graft materials. Because the harm is especially mechanical (about 50% lack of properties) the enhancement of mechanical properties will provide an oblique safety. Within the examine,²⁷ bovine and human patella tendons have been incubated with GP for durations as much as 12 h. Whereas YM elevated 2.4-fold within the bovine tendons, there was no important impact of crosslinking within the human tendons. The rise in elastic moduli within the gamma-irradiated tendons have been statistically insignificant each in animal and human specimens. It was surmised that the advantages of GP in human allografts might rely upon particular growing old facets.

Presently, the exogenous crosslinking of connective tissues utilizing chemical crosslinking brokers is considered a promising technique to deal with injured tissues, tendons amongst them, and GP as a most popular agent thanks primarily to its low cytotoxicity and efficient efficiency. Very just lately, a system for the sustained native supply of GP has been developed²⁸ by von Recum et al at Case Western Reserve College. They proposed cyclodextrins as carriers, and in contrast them with non-cyclic dextran. The cyclodextrins are molecules that possess cavities, and it was anticipated that the comparatively small molecule of GP might show molecular affinity for sure cavity sizes. The investigators demonstrated the sustained launch of GP from β-cyclodextrin, and its lively participation to the crosslinking response within the rat-tail tendons. Mechanical testing confirmed statistically important enhancements of sure final properties (power, vitality, and pressure to failure) following incubation with GP-cyclodextrin composites. The tendons handled with GP-dextrin (no cavities) confirmed much less mechanical augmentation, however nonetheless superior to the controls (empty β-cyclodextrin cavities, no GP). It was additionally discovered that GP improved resistance to collagenase. A cytotoxicity assay indicated that the protecting results have to be balanced with a sure stage of acceptable cytotoxicity.

Outcomes of Chemical Crosslinking

Desk 1 incorporates a synopsis of the outcomes printed to this point on the exogenous crosslinking of tendons utilizing chemical crosslinking brokers. Solely knowledge supported by statistical significance are included, and solely these outcomes are offered that confirmed precise mechanical augmentation of the tendinous tissue subjected to crosslinking. For uniformity, the outcomes have been re-calculated into share enhancement of the respective mechanical property.

Desk 1 Exogenous Crosslinking of Tendons Utilizing Chemical Brokers: a Selective Abstract of Printed Outcomes

Photochemical Crosslinking of Tendons

A perfect and protected process to realize exogenous crosslinking in dwelling connective tissues shall keep away from using chemical compounds. Even GP itself shows a sure stage of toxicity, and a compromise needs to be normally secured. As proven in our introduction, the UV-A radiation proved to be efficient and protected in treating corneal issues and in different medical procedures at present at varied investigative phases of growth. UV-A radiation might be delivered conveniently and the one chemical compounds concerned are innocuous photoinitiators resembling vitamin B2 (riboflavin). The technology of crosslinks by photochemistry is mediated by free radicals, that are sufficiently reactive as to instantly contain the amino residues of collagen chains, and due to this fact no extraneous chemical buildings are integrated within the crosslinkages.

Apparently, there are current studies on the reinforcement of musculoskeletal tissues completely different from tendons utilizing photochemically induced exogenous crosslinking. Vasilikos et al at Ulm College in Germany have investigated the photochemical crosslinking of intervertebral disc (IVD) using the system UV-A radiation/riboflavin, at an irradiance of three mW/cm². They’ve proven29 that the photocrosslinking of IVD led to lowered annular delamination, enhance of the peel power, and a major enhance of Younger’s modulus. In one other examine,³⁰ the topic was the shoulder’s glenohumeral joint capsule; its laxity is likely one of the essential lesions resulting in shoulder instability. Human capsular specimens (~1 mm³ every) have been harvested throughout shoulder surgical procedure, and uncovered to UV-A (365 nm) for half-hour after incubation in riboflavin. There was a transparent enhancement of the Younger’s modulus. On account of very small pattern measurement, the authors had to make use of AFM so as to measure the moduli. UV-A irradiation elevated capsule stiffness with out affecting structural group or inducing cell loss of life.

The usage of UV areas completely different from UV-A have been reported for crosslinking a tendon, nonetheless. For causes not clearly defined, collagen remoted from the rat-tail tendon was uncovered to UV-C radiation (wavelength 254 nm, vitality 4.9 eV) at an irradiance of 4.4 mW/cm² for very lengthy durations (2–8 hours).³¹ Such radiation is related to a photon vitality greater than that of UV-A (365 nm, vitality 3.4 eV), and is thought to be dangerous to organic matter, particularly over a prolonged publicity. We suppose that the examine was associated to the sterilization with UV-C of collagen-based implant supplies. As anticipated, the consequences of irradiation on mechanical properties have been detrimental, as the final word power, final pressure, and YM all decreased. Contemplating the hazard of irradiating dwelling tissue with UV-C rays, such research are irrelevant for our assessment, even when some have episodically reported mechanical augmentation.³²

In an earlier examine,³³ rat-tail tendon specimens have been irradiated with both seen gentle (> 370 nm) or UV (> 295 nm) areas within the presence of methylene blue as a photosensitizer. Chemical evaluation of the merchandise prompt that collagen crosslinking processes came about. Diffraction research confirmed that the photo-induced crosslinking didn’t have an effect on the molecular packing association of the native collagen. Nonetheless, this examine didn’t examine the impact on the mechanical properties.

So far, Snedeker’s group have printed¹⁹ the one report on investigating the exogenous crosslinking of tendinous collagen by publicity to UV-A radiation aiming at a medical software, in a examine the place two chemical crosslinking brokers (MG and GP) have been additionally included for comparability, as aforementioned. Strips of both RTTF or ESDFT have been soaked in diluted aqueous riboflavin for five minutes after which every uncovered to UV-A radiation (wavelength 375 nm) at an irradiance of 4.4 mW/cm² for half-hour. The radiation was generated by a UV diode supply and delivered at a distance of two.5 cm from the samples. Whereas the irradiated rat-tail tendon specimens confirmed enhanced mechanical properties (yield/final power and pressure, stiffness, and toughness), just like and even greater than these induced by GP, the UV irradiation didn’t have any impact of the properties of the equine tendon strips. GP was the one agent that induced mechanical enhancement in each sorts of tendons. It’s possible that for irradiation of the equine tendon a better irradiance ought to have been employed.

Dialogue

Tendons can turn out to be lax (unfastened) in two conditions: over time resulting from repetitive or extreme pressure (related to overuse, or with poor biomechanics), or instantly resulting from trauma. A part of their elasticity is misplaced when stretched past their regular capability, due to this fact the lax tendons don’t recoil to the preliminary size after the applying of pressure is stopped. Maybe a extra appropriate time period for a lax tendon could be stretched or elongated. Along with ligamentous laxity, the tendinous laxity contributes to instability within the joints, which will increase the danger for everlasting practical impairment.

It’s disputable whether or not the factitious exogenous crosslinking may very well be efficient in regenerating an overstretched and completely elongated tendon, whereas not but ruptured. In precept, the mechanical stabilization of the tissue might be achieved by crosslinking, which can arrest the power for additional stretching. The crosslinking can’t obtain, nonetheless, the restoration of the preliminary contractility stage, ie, the shortening again to the preliminary size. This could occur solely in two conditions: (a) by way of the actin-mediated exercise of the tenocytes that may generate traction forces in opposition to an extracellular matrix capable of contract the lax tendons; and (b) if the collagen crosslinking response as such has a contractile impact (shrinkage) on the collagen. The previous subject (a) is said with the tendon laxity ensuing from lack of collagen rigidity and disrupting cytoskeletal tensional homeostasis in tenocytes, and in addition inducing catabolic adjustments resembling upregulation of collagenase expression and apoptosis. Nonetheless, an actin-based cell contraction mechanism can re-establish the tensional homeostasis within the lax tendons. This mechanism has been extensively studied by Arnoczky’s group at Michigan State College.^34–36 Nonetheless, we have no idea whether or not it will likely be lively in a tendon beforehand subjected to the method of chemical or photochemical exogenous crosslinking, and due to this fact affected by sure structural modifications.

When contemplating the latter subject (b), we’re not conscious of any reported proof that the crosslinking of collagen is related to the shrinkage of its matrix, which might allow a contractile impact on stretched connective tissues. In any other case, it’s well-known that the publicity of collagen basically, and tendinous collagen particularly, to temperatures exceeding ~60°C, results in its shrinkage,^37–41 a course of that has really been prompt as a remedy for glenohumeral instability, based mostly on experiments with human joint capsule or bovine extensor tendons at temperatures over 60°C.^42,43 On one other word, the hyperthermic remedy for tendinopathies, which is carried out at temperatures round 42°C, ends in enhanced contraction charges within the lax tendons, however appears to be based mostly on a shrinking mechanism not but elucidated.^44,45 Such thermal shrinking processes are bodily in nature and should not associated to chemical reactions resulting in crosslinking.

Taking a look at present and proposed strategies of remedy for tendinopathies, and on the restore methods based mostly on bioengineering (eg, biomaterials, tissue engineering, and cell-based therapies), as offered in various seminal opinions,^46–51 one can see {that a} methodology based mostly on collagen crosslinking has by no means been talked about as a viable different. This clearly implies that there’s a want for extra experimental work to be accomplished for elevating the official curiosity of orthopedic specialists.

Conclusion

There’s not a lot curiosity at present in growing therapies for tendinopathies based mostly on the exogenous crosslinking of tendon collagen, though topical studies emerge episodically from some analysis facilities. Sure studies have offered proof for the mechanical augmentation of crosslinked tendons, however in lots of instances the outcomes could also be seen as tenuous. Additional analysis work is clearly wanted so as to develop such therapies. On the subject of security, it’s value contemplating the alternative of chemical crosslinking with UV radiation-induced crosslinking.