**5. Cellular approaches**

The potential of stem cells for regenerative medicine and for tissue engineering applications has been reported many times with convincing evidence *in vitro* and *in vivo* and comprehensive information given in recent review articles [12, 95]. Hence, although tenocytes would be the first and self-evident cell source to be used in tendon tissue engineering [20, 96, 97], there are more reports based on stem cells for the same purpose. For example, adipose-derived stem cells were seeded onto biphasic silk scaffold in order to fabricate a tendon-to-bone interface, mimicking the gradient-like structure of the enthesis [57]. Adipose-derived stem cells are easily harvested and differentiated toward a desired lineage [98] and amounts per gram of tissue are higher as compared to other stem cells sources like bone marrow [99]. Hence, these cells are very well suited for tissue engineering purposes, like tendon tissue engineering [100, 101]. Among different stem cell sources, however, the best source of stem cells for tendon tissue engineering is reported to be tendon stem cells, although their availability is limited and the harvesting protocol everything else than easy [32]. An interesting study reports the beneficial effect of seeding tendon-derived stem cells onto a chitosan-β-glycerophosphate-collagen hydrogel scaffold intended to repair an Achilles tendon defect in a rat model [74]. The healing was enhanced as indicated by the improvement in histological and immunohistochemical outcomes. In addition, the increase in the biomechanical properties of the regenerated tissue at both 4 and 6 weeks post-operation also supported the effectiveness of tendon-derived stem cells [74].

The *in vitro* preparation of cell-based TECs highly determines the mechanical properties; cellseeded scaffolds cultivated under static conditions have different characteristics compared to TECs cultivated under dynamic conditions—as for example cultivation in a bioreactor with medium perfusion flow and/or tensile stretching/compression regimen [102]. Collagen sponges seeded with MSCs have been reported to have significantly higher mechanical properties when cultivated with mechanical stimulation than under static conditions [103]. Also, the expression of collagen I and III are increased upon mechanical stimulation, as shown for rabbit MSC/collagen sponges and murine MSC/collagen sponges [104]. In such approaches of dynamic cell culture and cell-seeded TECs, physical experimental parameters like frequency, amplitude, medium flow rate, etc., have to be carefully tuned in order to get the desired differentiation (if stem cell-based) and the intended biomechanics [105–108].

Other concepts in tendon tissue engineering are based on decellularization of a natural xenograft in order to avoid immunorejection [4, 109–111] or decellularization of a primarily cellseeded construct in order to generate a scaffold coated with the components of the ECM of a certain cell type (instructive ECM) cultivated under well-defined conditions [112, 113]. Such decellularized graft materials can be applied in daily clinical practice more easily than cellseeded TECs; because storage is facilitated without (living) cells [114].

Furthermore, there are gene therapy strategies including adeno-associated viral type 2 vector (AAV2) and micro-RNA related gene therapy aiming at improving strength of the repaired tendon as well as decreasing adhesion formation to the surrounding tissue [115, 116]. Moreover, some approaches deal with delivering certain (growth) factors, supporting the regeneration process of tendons [117, 118].

**5. Cellular approaches**

the tube is pulled over (B).

168 Tissue Regeneration

The potential of stem cells for regenerative medicine and for tissue engineering applications has been reported many times with convincing evidence *in vitro* and *in vivo* and comprehensive information given in recent review articles [12, 95]. Hence, although tenocytes would be the first and self-evident cell source to be used in tendon tissue engineering [20, 96, 97], there are more reports based on stem cells for the same purpose. For example, adipose-derived stem cells were seeded onto biphasic silk scaffold in order to fabricate a tendon-to-bone interface, mimicking the gradient-like structure of the enthesis [57]. Adipose-derived stem cells are easily harvested and differentiated toward a desired lineage [98] and amounts per gram of tissue are higher as compared to other stem cells sources like bone marrow [99]. Hence, these cells are very well suited for tissue engineering purposes, like tendon tissue engineering [100, 101]. Among different stem cell sources, however, the best source of stem cells for tendon tissue engineering is reported to be tendon stem cells, although their availability is limited and the harvesting protocol everything else than easy [32]. An interesting study reports the beneficial effect of seeding tendon-derived stem cells onto a chitosan-β-glycerophosphate-collagen hydrogel scaffold intended to repair an Achilles tendon defect in a rat model [74]. The healing was enhanced as indicated by the improvement in histological and immunohistochemical outcomes. In addition, the increase in the biomechanical properties of the regenerated tissue at both 4 and 6 weeks

**Figure 2.** Application of DegraPol®. An electrospun DegraPol® tube is placed around a fully transected rabbit Achilles tendon (A) in order to deliver a growth factor to the repair site. The laceration is sutured by a 4-strand Becker suture and

post-operation also supported the effectiveness of tendon-derived stem cells [74].

The *in vitro* preparation of cell-based TECs highly determines the mechanical properties; cellseeded scaffolds cultivated under static conditions have different characteristics compared to TECs cultivated under dynamic conditions—as for example cultivation in a bioreactor with medium perfusion flow and/or tensile stretching/compression regimen [102]. Collagen sponges seeded with MSCs have been reported to have significantly higher mechanical properties when cultivated with mechanical stimulation than under static conditions [103]. Also, the expression of collagen I and III are increased upon mechanical stimulation, as shown for rabbit MSC/collagen sponges and murine MSC/collagen sponges [104]. In such approaches of TGF-β1 plays an important role during tendon healing and has an influence on adhesion formation, an unwanted side effect during tendon healing. Therefore, regulation of TGF-β1 through application of micro-RNA specifically inhibiting the function of TGF-β1 was tested in a chicken flexor tendon model [119]; TGF-β protein expression in the tendons decreased on increasing the vector dosage. As a consequence, the adhesion extent significantly decreased 6 and 8 weeks post-injury; however, tendon strength unfortunately was also reduced [119]. Another study showed that gene therapy to produce supernormal amounts of bFGF or VEGF supported the intrinsic tendon healing in a chicken flexor tendon model—with a significantly higher tendon strength by 68–91% from week 2 after AAV2-bFGF treatment and by 82–210% from week 3 after AAV2-VEGF compared to controls [120]. At the same time, adhesion formation was not adversely affected.

Because decorin and IL-10 downregulate TGF-β1, another approach included co-delivery of decorin and IL-10 transgenes from a collagen hydrogel system to a tenocyte culture *in vitro*. As expected, TGF-β1 was downregulated and simultaneously also collagen I and III and fibronectin. The authors concluded that this approach might be a useful tool against scar formation (extensive fibrosis), the system has not yet been tested *in vivo*, however [121]. Moreover, another AAV-based approach was the delivery of VEGF to chicken flexor tendons; after complete transection of these tendons, 2 × 10<sup>9</sup> particles of AAV2-VEGF or saline (as control) were injected before they were surgically repaired [122]. The outcome was a significantly increased ultimate strength 4, 6 and 8 weeks post-operation, while the adhesion was unaffected [122]. Hence, such gene therapy approaches might get more significance also in daily clinical life, as they are easily performed (injection of a small volume only) and show promising effects.

Another nice example has been shown using Scx-transduced tendon-derived stem cells in a rat unilateral patellar tendon window injury model. For transplantation, a TEC based on fibrin and transduced cells was used. Tendon repair was significantly improved in terms of histology and biomechanics *in vivo*. *In vitro* results showed that Scx-transduced tendonderived stem cells expressed tendon- and also cartilage-related genes to a higher level; as for osteogenic markers (which might be regarded as an unwanted differentiation), the results remained inconclusive [123]. Further reports on gene therapy dealing with the tendon-togone interface also used BMPs [124].

**Author details**

Johanna Buschmann

Zurich, Switzerland

2006;**12**:369-379

560455246676

jse.2011.10.021

2013;**24**:211-220. DOI: 10.1007/s10856-012-4791-3

**References**

Address all correspondence to: johanna.buschmann@usz.ch

Clinics. 2013;**29**:191-206. DOI: 10.1016/j.hcl.2013.03.001

Orthopaedicae et Traumatologiae Cechoslovaca. 2014;**81**:371-379

Plastic Surgery and Hand Surgery, Department of Surgical Research, University Hospital

Tissue Engineering of Tendons

171

http://dx.doi.org/10.5772/intechopen.73507

[1] Elliot D, Giesen T. Primary flexor tendon surgery: The search for a perfect result. Hand

[2] Vališ P, Sklenský J, Repko M, Rouchal M, Novák J, Otaševič T. Most frequent causes of autologous graft failure in anterior cruciate ligament replacement. Acta Chirurgiae

[3] Karabekmez F, Zhao C. Surface treatment of flexor tendon autograft and allograft decreases adhesion without an effect of graft cellularity: A pilot study. Clinical Orthopaedics and Related Research®. 2012;**470**:2522-2527. DOI: 10.1007/s11999-012-2437-x [4] Lui H, Vaquette C, Bindra R. Tissue engineering in hand and surgery: A technology update. Journal of Hand Surgery-American Volume. 2017;**42**:727-735. DOI: 10.1016/j.jhsa.2017.06.014 [5] Juncosa-Melvin N, Boivin GP, Gooch C, Galloway MT, West JR, Dunn MG, et al. The effect of autologous mesenchymal stem cells on the biomechanics and histology of gelcollagen sponge constructs used for rabbit patellar tendon repair. Tissue Engineering.

[6] Vaquette C, Slimani S, Kahn CJ, Tran N, Rahouadj R, Wang X. A poly(lactic-co-glycolic acid) knitted scaffold for tendon tissue engineering: An in vitro and in vivo study. Journal of Biomaterials Science. Polymer Edition. 2010;**21**:1737-1760. DOI: 10.1163/092050609x12

[7] Beason DP, Connizzo BK, Dourte LM, Mauck RL, Soslowsky LJ, Steinberg DR, et al. Fiberaligned polymer scaffolds for rotator cuff repair in a rat model. Journal of Shoulder and Elbow Surgery/American Shoulder and Elbow Surgeons. 2012;**21**:245-250. DOI: 10.1016/j.

[8] Pietschmann MF, Frankewycz B, Schmitz P, Docheva D, Sievers B, Jansson V, et al. Comparison of tenocytes and mesenchymal stem cells seeded on biodegradable scaffolds in a full-size tendon defect model. Journal of Materials Science-Materials in Medicine.

[9] Dines JS, Weber L, Razzano P, Prajapati R, Timmer M, Bowman S, et al. The effect of growth differentiation factor-5-coated sutures on tendon repair in a rat model. Journal of

Shoulder and Elbow Surgery. 2007;**16**:S215-SS21. DOI: 10.1016/j.jse.2007.03.001
