Jin Bo Tang

Tendon Healing In Vivo: Gene Expression and Production of Multiple Growth Factors in Early Tendon Healing Period

PURPOSE The actions of growth factors during healing of injured flexor tendons are not well characterized, although information pertinent to some individual growth factors is available. We studied gene expression and protein production of a number of growth factors at several time points during the early healing period in a chicken model. METHODS Seventy-four long toes of 37 white Leghorn chickens were used. The flexor digitorum profundus tendons of 60 toes were surgically repaired after complete transection and were harvested for analysis 3, 5, 7, 9, 14, and 21 days after surgery. The expression of 6 growth factors was studied at 4 time points after surgery with real-time quantitative polymerase chain reactions, and production and distribution of 3 growth factors at all 6 time points were studied by immunohistochemical staining with antibodies. Fourteen tendons that had no surgery served as day 0 controls. Tendon healing status was also assessed histologically. RESULTS Throughout the early tendon healing period, connective tissue growth factor (CTGF) and transforming growth factor beta (TGF-beta) showed high levels of gene expression. Levels of gene expression of vascular endothelial growth factor (VEGF) and insulin-like growth factor 1 (IGF-1) were high or moderately high. Expression of the TGF-beta gene was upregulated after injury, whereas the basic fibroblast growth factor (bFGF) gene was downregulated at all postsurgical time points and expressed at the lowest levels among 6 growth factor genes 2 to 3 weeks after surgery. The platelet-derived growth factor B (PDGF-B) gene was also minimally expressed. Findings of immunohistochemistry corresponded to TGF-beta, bFGF, and IGF-1 gene expression. CONCLUSIONS In this model, up to 3 weeks after surgery, gene expression and production of TGF-beta are high and are upregulated in this healing period. However, expression of the bFGF gene and protein is low and decreases in the healing tendon. The CTGF, VEGF, and IGF-1 genes are expressed at high or moderately high levels, but PDGF-B is minimally expressed.

Enhanced peripheral nerve regeneration through a poled bioresorbable poly(lactic-co-glycolic acid) guidance channel

In this study we investigated the effects of materials prepared with electrical poling on neurite outgrowth in vitro and nerve regeneration in vivo. Neuro-2a cells were seeded on poled and unpoled poly(lactic-co-glycolic) (PLGA) films and observed at time periods 24, 48 and 72 h post-seeding. The percentage of cells with neurites and the neurites per cell were quantified using light microscopy. At 48 and 72 h post-seeding, both the number of cells with neurites and the neurites per cell were significantly increased on the poled films compared to those on unpoled films. An established rat sciatic nerve model was used for in vivo studies to assess the effects of PLGA guides, poled for two different periods, on peripheral nerve regeneration. Guides were inserted in rats to bridge a 1.0 cm gap created in the right sciatic nerve. After four weeks, nerves regenerated through poled guides displayed a significant increase in conduction velocity and significantly increased numbers of axons across the guides, as compared to nerves regenerating through an unpoled guidance channel. Electrical poling was shown to promote neurite growth, axon regeneration and the conduction rate of the repaired nerve. We concluded that guides prepared with electrical poling enhance peripheral nerve regeneration.

PDGF gene therapy enhances expression of VEGF and bFGF genes and activates the NF-kappaB gene in signal pathways in ischemic flaps.

Background: Gene therapy is a novel approach for enhancing the viability of ischemic flaps. Expression of growth factor genes pertinent to angiogenesis and activation of genes of relevant signal pathways are imperative for improving flap viability. The authors investigated the gene expression profiles of growth factors and signal transduction pathways in ischemic flaps after PDGF gene therapy. Methods: Twenty Sprague-Dawley rats were divided into two groups. The experimental group (n = 10) received the plasmid vector containing the PDGF cDNA injected into the dermis of the flap area, whereas the control group (n = 10) received the physiologic saline. Seven days later, a dorsal random flap was raised. Seven days after surgery, flap viability was assessed, and expression of VEGF, bFGF, TGF-&bgr;1, NF-&kgr;B, Erk2, Stat1, and Smad2 genes of the NF-&kgr;B, MAPK, JAK-STAT, and Smad pathways was assessed by quantitative analysis of the products of reverse-transcriptase polymerase chain reaction. Results: Transfer of exogenous PDGF gene significantly improved flap viability (p = 0.011). Levels of expression of VEGF and bFGF genes in the flap were significantly elevated after PDGF gene transfer (p = 0.0001 and p = 0.001, respectively). Expression of the NF-&kgr;B gene was significantly elevated (p = 0.041). In contrast, expression of TGF-&bgr;1, and Erk2, Stat1, and Smad2 genes was not changed. Conclusions: Transfer of exogenous PDGF gene to ischemic flaps promotes expression of VEGF and bFGF genes and activation of NF-&kgr;B gene in addition to its effects on the PDGF gene. The finding implies that transfer of the gene of one growth factor ultimately improves the expression of the genes of multiple growth factors. Activation of the NF-&kgr;B gene suggests that the NF-&kgr;B pathway may be important in enhancement of flap viability and will likely be a target of future efforts of regulation of signaling process in treatment of ischemic flaps.

Effect of A2 Pulley Release on Repaired Tendon Gliding Resistance and Rupture in a Chicken Model

PURPOSE Release of part of the A2 pulley has been suggested to improve function after zone II primary flexor tendon repairs. The purpose of this study was to measure the effect of A2 pulley release compared with pulley preservation on repaired tendon gliding resistance and rupture in the early postoperative period in a chicken model. METHODS We divided 104 long toes from 52 Leghorn chickens into 2 experimental groups. In group 1, the flexor digitorum profundus tendons were cut completely and repaired surgically with either release or preservation of the A2 pulley. The tendon laceration was positioned so that the repair had to glide through the pulley with toe flexion. The toes were subjected to simulated active flexion at the end of the 2nd and 4th postsurgical weeks. The rupture rates of the tendons were recorded. In group 2, the volar two thirds of the tendons were cut and repaired, also in the region of the A2 pulley, with the pulley released or preserved. The force resisting tendon motion and the work of digital flexion with simulated active digital flexion were tested 3, 5, 7, and 14 days after surgery. RESULTS At the end of the 2nd and 4th weeks, the tendon repairs showed significantly higher rates of rupture in the presence of an intact A2 pulley compared with those with the pulley released. Tendon gliding resistance was significantly increased with preservation of the pulley on postsurgical days 3, 5, 7, and 14; these toes required a greater number of motion cycles to reach steady levels of resistance and showed significantly higher steady resistance after digital motion. CONCLUSIONS Release of the A2 pulley decreases the rate of repair rupture and the resistance to tendon gliding when the site of tendon repair has to glide through the pulley in the early postsurgical period. The findings of this study support partial release of the A2 pulley to favor successful tendon repair.

Tendon Healing In Vitro: Adeno-associated Virus-2 Effectively Transduces Intrasynovial Tenocytes with Persistent Expression of the Transgene, but Other Serotypes Do Not

Background: Transfer of exogenous growth factor genes to injured tendons offers a promising method for strengthening tendon repairs. Adeno-associated virus vectors have advantages of being both nonpathogenic and nontoxic. The authors explored the efficiency of transduction of intrasynovial tenocytes with different serotypes of adeno-associated virus (AAV) and the persistency of its expression of a growth factor transgene. Methods: Tenocytes were obtained from cultures of rat intrasynovial tendons and distributed to 82 wells in eight culture plates and to 30 culture dishes. The tenocytes in the wells were treated with AAV1, AAV2, AAV3, AAV4, AAV5, AAV7, and AAV8 vectors containing the lacZ gene, and plasmid vectors (pCMVβ-lacZ). The tenocytes were stained with in situ β-galactosidase 5 days later. The basic fibroblast growth factor (bFGF) gene was cloned to the AAV2 vector to construct the AAV2-bFGF vector, which transduced tenocytes in culture dishes. Expression of the transgene was measured over 3 weeks and analyzed statistically. Results: AAV2 effectively delivered exogenous genes to proliferating intrasynovial tenocytes. In contrast, other tested adeno-associated viruses transduced tenocytes minimally or not at all. The efficiency of gene transfer by AAV2, indicated by the percentage of cells with positive β-galactosidase staining, was significantly greater than that by a plasmid vector (p = 0.001). Expression of the bFGF gene in tenocytes transduced with the AAV2-bFGF was significantly higher than that in the control over the 3-week period (p < 0.01). Conclusions: Gene transfer to tenocytes by AAV2 is more efficient than that by a plasmid vector. However, other adeno-associated virus serotypes cannot effectively transduce tenocytes. The bFGF gene can be delivered to intrasynovial tenocytes by the AAV2 vector effectively, and the gene transfer significantly increases expression of bFGF gene over 3 weeks.

Basic FGF or VEGF gene therapy corrects insufficiency in the intrinsic healing capacity of tendons

Tendon injury during limb motion is common. Damaged tendons heal poorly and frequently undergo unpredictable ruptures or impaired motion due to insufficient innate healing capacity. By basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) gene therapy via adeno-associated viral type-2 (AAV2) vector to produce supernormal amount of bFGF or VEGF intrinsically in the tendon, we effectively corrected the insufficiency of the tendon healing capacity. This therapeutic approach (1) resulted in substantial amelioration of the low growth factor activity with significant increases in bFGF or VEGF from weeks 4 to 6 in the treated tendons (p < 0.05 or p < 0.01), (2) significantly promoted production of type I collagen and other extracellular molecules (p < 0.01) and accelerated cellular proliferation, and (3) significantly increased tendon strength by 68–91% from week 2 after AAV2-bFGF treatment and by 82–210% from week 3 after AAV2-VEGF compared with that of the controls (p < 0.05 or p < 0.01). Moreover, the transgene expression dissipated after healing was complete. These findings show that the gene transfers provide an optimistic solution to the insufficiencies of the intrinsic healing capacity of the tendon and offers an effective therapeutic possibility for patients with tendon disunion.

Cellular Apoptosis and Proliferation in the Middle and Late Intrasynovial Tendon Healing Periods

PURPOSE Cellular apoptosis might be an important molecular event in the middle or late healing periods of intrasynovial tendons, but this has not been studied. We aimed to investigate cellular apoptosis and corresponding cellular proliferation in the middle and late healing stages of intrasynovial tendons. METHODS The flexor digitorum profundus tendons of 48 long toes (24 chickens) were completely transected within the sheath region and were repaired surgically. At days 28, 42, 56, and 84 after surgery, tendons were harvested and sectioned. In situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to detect apoptotic cells. The sections were stained immunofluorescently with antibodies to proliferating cell nuclear antigen to assess proliferation and to Bcl-2 (an anti-apoptotic protein). Positively stained tenocytes were counted, and their distributional differences were verified in 3-dimensional images. RESULTS The repaired intrasynovial tendons exhibited generally greater apoptosis in the surface region than in the core. The differences were more remarkable in the extended region than in the junction region of the cut tendon. At the core of the junction site, apoptosis of tenocytes was pronounced at all time points, but it was less severe at the core of the extended region. The proliferating cell nuclear antigen-positive and Bcl-2-positive tenocytes decreased significantly and continually at days 28, 42, and 56, respectively; these tenocytes were at a minimum at days 56 and 84. CONCLUSIONS Apoptotic changes of tenocytes are most marked in the surface region and in the junction region of the healing tendon in the middle and late healing stages. Apoptosis in the core is less dramatic compared to that in the surface in the extended tendon regions. Cellular proliferation declines drastically and is minimal at days 56 and 84. CLINICAL RELEVANCE Tenocyte apoptosis in the middle and late stages might be an important event contributing to intrasynovial tendon remodeling, which affects the healing strength and formation of adhesions.

New Tendon, Nerve, and Bone Surgical Methods Impacting the Practice of Hand Surgery

This new concept of hand surgery was advocated by Dr Donald Lalonde and colleagues in 2007. Dr Lalonde has performed over 2000 surgeries using this approach—eliminating anesthesiainduced complications—and reports high clinical safety rates. In just a few years, we now see widespread popularization of this method in many countries. This has been an important and wellintegrated surgical method in hand surgery. The anesthesia is achieved by injecting 1% lidocaine with 1:100,000 epinephrine in the hand (or 0.5% lidocaine with 1:200,000 epinephrine when 50 to 100 mL is injected; see refs.). The following are recognized fields utilizing this technique. Tendon transfer surgery is most benefited from this approach. Proper tension of the transferred tendon can be judged and adjusted by prompting the patient to actively move his or her digit intraoperatively. This approach works well in almost all tendon transfers, including the transfer of the extensor indicis proprius to the extensor pollicis longus tendon, the flexor digitorum superficialis of the ring finger to the flexor pollicis longus or abductor pollicis brevis, and so on. Primary flexor and extensor tendon repairs are also ideal forwide-awakesurgery.Gappingand tension at the repair site can be assessed under direct visualization on the operating table with the patient actively flexing or extending his or her digit. This technique allows for the most reliable judgment of tendon repair that can safely proceed to early active motion. A surgeon should ensure the absence of gapping between tendon ends under active digital motion before leaving the operating room. Carpal tunnel release, cubital tunnel release, and release of compression of the anterior or posterior interosseous nerves can also be performed under just local anesthesia. In the operating room, this

Re: Brockardt CJ, Sullivan LG, Watkins BE et al. Evaluation of simple and looped suture and new material for flexor tendon repair. J Hand Surg Eur. 2009, 34: 329–32:

We read with interest this paper about the use of simple and looped sutures in repairing tendons in an experimental setting. The authors found that their method B (a four-strand Kessler repair, using the needle leading double strands) did not provide the strength as much as their method C (the double Kessler repair, using the needle leading one strand). Based on this finding, they considered that the looped suture does not provide the same strength as two separate throws; the authors pointed out that these appear different from the findings of Cao and Tang (2005) using a looped suture for a modified Tang repair. We have tried to read the details of how they tied the knots, and whether they incorporated any locking anchors into the tendon–suture junction. We found no clear information about how the knots were made in the text, except in the drawings in Fig 1. We saw no locking anchors to the tendons based on the suture’s loop design. It appears to us that in method B, the authors led two strands which were looped together through the tendon, without taking advantages of the suture’s loops to lock the tendon substance. If this is true, the authors’ repair could be achieved just as easily by using a single needle attached to two independent, rather than looped, strands. The advantages of the loop have not been utilized for making tendon–suture junctions. In other words, the suture’s loop design is not essential for that repair method. One needle carrying two independent strands can accomplish the same repair. Our experience with looped sutures is that their advantage is in the ability to lock anchors to the tendon, and the possibility of leading two strands into the tendon at one pass (Cao and Tang, 2005; Tang 2007). The greater holding power is generated by their locking junctions with the tendon. We have used the needle carrying two independent strands in repairing the tendons, achieving a repair configuration identical to the method B and reported at the 10th IFSSH meeting in 2007. In comparing a four-strand Tang method using a looped suture, and incorporating the locking anchors, a method of four-strand Kessler repair (using one needle carrying two independent strands), and a double Kessler method, we found that the four-strand Kessler repair was weaker than the other two. Our and the authors’ reports actually present similar findings – without this locking type of tendon–suture junction using the loop, the repair using one needle carrying two strands is weak, regardless of the four strands across the repair site. The four-strand Kessler repair, achieved both in our report and in the study of Brockardt et al. (2009), differs distinctly from the four-strand Tang and double Kessler methods in biomechanical properties. The findings – by the authors regarding a repair without taking advantage of the essential merits of a loop suture – cannot be extrapolated to those methods that were accomplished with locking anchors in the tendon–suture junctions using the loops, a key component of the repair to repair strength.

Application of AAV2-Mediated bFGF Gene Therapy on Survival of Ischemic Flaps : Effects of Timing of Gene Transfer

Necrosis of surgically transferred flaps is a major problem in reconstructive surgery. We investigated efficacy of a new vector system—adeno-associated viral 2 (AAV2)-mediated bFGF gene transfer to enhance survival of the ischemic flap. Thirty-eight Sprague-Dawley rats were divided into 3 gene therapy groups and 1 nontreated control of 9 or 10 each. 7.5 × 1010 AAV2-bFGF viral particles were injected to the dorsum of each of the 29 rats; these rats were divided into 3 groups according to the timing of flap elevation. At the time of surgery, 1 week, and 2 weeks after surgery, flaps of 3 × 7 cm were raised. One week after surgery, flap viability was measured. Vascularization and immunohistochemical staining of the bFGF were evaluated of histologic sections. Flap viability was significantly improved by the AAV2-bFGF gene therapy at the time of surgery, and the flaps with the greatest survival area were found in the rats injected with AAV2-bFGF, 2 weeks before surgery. However, flap viability was significantly decreased by the gene therapy 1 week before surgery. Histologically, vascularity was increased in the groups with AAV2-bFGF injection and immunohistochemical staining showed greatly enhanced bFGF expression by gene transfer. The novel approach of AAV2-bFGF gene therapy shows encouraging manifestations in improving survival of flaps when the flaps are prefabricated during or 2 weeks before surgery.