Hypoxia as a stimulus for tissue formation: The concept of organogenesis in microsurgically vascularized tissue engineering constructs.

Axial vascularization of tissue constructs is essential to maintain an adequate blood supply for a stable regeneration of a clinically relevant tissue size. The versatility of the arterio-venous loop (AVL) has been previously shown in various small and large animal models as well as in clinical reports for bone regeneration. We have previously demonstrated the capability of the AVL to induce axial vascularization and to support the nourishment of tissue constructs in small animal models after applying high doses of ionizing radiation comparable to those applied for adjuvant radiotherapy after head and neck cancer. We hypothesize that this robust ability to induce regeneration after irradiation could be related to a state of hypoxia inside the constructs that triggers the HIF1 (hypoxia induced factor 1) - SDF1 (stromal derived factor 1) axis leading to chemotaxis of progenitor cells and induction of tissue regeneration and vascularization. We analyzed the expression of HIF1 and SDF1 via immunofluorescence in axially vascularized bone tissue engineering constructs in Lewis rats 2 and 5 weeks after local irradiation with 9Gy or 15Gy. We also analyzed the expression of various genes for osteogenic differentiation (collagen 1, RUNX, alkaline phosphatase and osteonectin) via real time PCR analysis. The expression of HIF1 and SDF1 was enhanced two weeks after irradiation with 15Gy in comparison to non-irradiated constructs. The expression of osteogenic markers was enhanced at the 5-weeks time point with significant results regarding collagen, alkaline phosphatase and osteonectin. These results indicate that the hypoxia within the AVL constructs together with an enhanced SDF1 expression probably play a role in promoting tissue differentiation. The process of tissue generation triggered by hypoxia in the vicinity of a definite vascular axis with enhanced tissue differentiation over time resembles hereby the well-known concept of organogenesis in fetal life.

Systemically injected bone marrow mononuclear cells specifically home to axially vascularized tissue engineering constructs.

Inducing axial vascularisation of tissue engineering constructs is a well-established method to support tissue growth in large 3-dimensional tissues. Progenitor cell chemotaxis towards axially vascularized tissues has not been well characterized. In a prospective randomized controlled study including 32 male syngeneic Lewis rats we investigated the capability of the axially vascularized constructs to attract systemically injected bone marrow mononuclear cells (BMMNCs). The underlying mechanism for cell homing was investigated focusing on the role of hypoxia and the SDF1-CXCR4-7 axis. Sixteen animals were used as donors for BMMNCs. The other animals were subjected to implantation of a tissue engineering construct in the subcutaneous groin region. These constructs were axially vascularized either via an arteriovenous loop (AVL, n = 6) or via uninterrupted flow-through vessels (non-AVL, n = 10). BMMNCs were labelled with quantum dots (Qdot® 655) and injected 12 days after surgery either via intra-arterial or intravenous routes. 2 days after cell injection, the animals were sacrificed and examined using fluorescence microscopy. The Qdot® 655 signals were detected exclusively in the liver, spleen, AVL constructs and to a minimal extent in the non-AVL constructs. A significant difference could be detected between the number of labelled cells in the AVL and non-AVL constructs with more cells detected in the AVL constructs specially in central zones (p <0.0001). The immunohistological analysis showed a significant increase in the absolute expression of HIF-1 in the AVL group in comparison to the non-AVL group. The PCR analysis confirmed a 1.4-fold increase in HIF-1 expression in AVL constructs. Although PCR analysis showed an enhanced expression of CXCR4 and CXCR7 in AVL constructs, no significant differences in SDF1 expression were detected via immunohistological or PCR analysis. At the examined time point, the AVL constructs can attract BMMNCs in a mechanism probably related to the hypoxia associated with a robust tissue formation.

Irradiation Delays Tissue Growth but Enhances Osteogenic Differentiation in Vascularized Constructs.

BACKGROUND: Regenerative medicine is still deficient in the reconstruction after cancer due to impaired vascularization after radiotherapy and due to the need to substitute larger defects after tumor excision. Aiming at introducing regenerative medicine for reconstruction after cancer, we tested an axially vascularized bone construct in an experimental setting that mimics the clinical situation after tumor resection and adjuvant radiotherapy. METHODS: Twenty bone constructs were axially vascularized using microsurgically created arteriovenous loops and were implanted subcutaneously in Lewis rats. After 2 weeks, the animals were randomly allocated either to receive a clinically relevant single dose of external beam irradiation or not (n = 10 for each group). The animals were sacrificed either after 1 week or 10 weeks after irradiation (n = 5 for each time point). The constructs were tested for vascularization, tissue growth, cellular proliferation, cellular apoptosis, and osteogenic differentiation via histomorphometric, immunohistochemical, and polymerase chain reaction (PCR) analysis. One construct per group was subjected at 10 weeks to qualitative micro-computed tomography (CT) imaging. RESULTS: Tissue generation and cellular proliferation were significantly reduced at 1 week after irradiation, but no longer significantly different after 10 weeks.No significant differences in vascularization were detected at any time point. Apoptosis did not show any statistically significant differences between both groups at both time points. At the late time point, mature bone was considerably more in the irradiated group, but the results were not statistically significant. PCR analysis showed a significantly enhanced expression of osteocalcin in the irradiated group at 1 week. Micro-CT imaging showed that both constructs were adequately vascularized with no evident morphologic differences regarding vascular density or vascular distribution. CONCLUSIONS: Axially vascularized bone constructs can withstand clinically relevant doses of irradiation and retain their angiogenic and osteogenic potential in the long term. Irradiation led to a delayed tissue generation with a comparatively enhanced osteogenic differentiation within the constructs.

Independent predisposing factors for subcutaneous and deep wound collection after total thyroidectomy, a prospective cohort study.

BACKGROUND: The literature contains diverse and sometimes contradicting results about wound seroma following thyroidectomy. This is probably due to the subjective clinical estimation of seroma, or due to failure to differentiate between the occurrence of subcutaneous (SC) and deep wound collections. This work aimed at objectively investigating the factors affecting subcutaneous and deep wound seroma after thyroidectomy. METHODS: The relation between various operative and clinico-pathological factors and the collection formation was prospectively analyzed in a cohort of 100 patients after conventional thyroidectomy. Wound seroma was assessed clinically and via high-resolution ultrasonography at 24 h, 48 h and two weeks postoperatively. Sonographically detected collections were expressed as SC and/or deep wound collections according to the relation to strap muscles. RESULTS: Operative duration was the only independent factor significantly affecting the incidence of clinical seroma. Older patients (>40ys) showed significantly larger volumes of early SC collections. Early postoperative pain was significantly related to drain insertion, to the occurrence of clinical seroma and to the volume of SC collections.Sonographically, suction drains and shorter operative durations resulted in significantly less amount of deep collections. Suction drains did not result in less amount of SC collections or in a lower incidence of clinical seroma. CONCLUSIONS: Operative duration is the only independent factor significantly related to clinically-detected postoperative seroma with its subsequent postoperative pain. Especially in elderly patients, a flapless technique would be recommended as these patients developed larger volumes of SC collections with subsequent higher pain scores, even if seroma was not clinically detected.

Axially vascularized tissue-engineered bone constructs retain their in vivo angiogenic and osteogenic capacity after high-dose irradiation.

In order to introduce bone tissue engineering to the field of oncological reconstruction, we are investigating for the first time the effect of various doses of ionizing irradiation on axially vascularized bone constructs. Synthetic bone constructs were created and implanted in 32 Lewis rats. Each construct was axially vascularized through an arteriovenous loop made by direct anastomosis of the saphenous vessels. After 2 weeks, the animals received ionizing irradiation of 9 Gy, 12 Gy and 15 Gy, and were accordingly classified to groups I, II and III, respectively. Group IV was not irradiated and acted as a control. Tissue generation, vascularity, cellular proliferation and apoptosis were investigated either 2 or 5 weeks after irradiation through micro-computed tomography, histomorphometry and real-time polymerase chain reaction (PCR). At 2 weeks after irradiation, tissue generation and central vascularity were significantly lower and apoptosis was significantly higher in groups II and III than group IV, but without signs of necrosis. Cellular proliferation was significantly lower in groups I and II. After 5 weeks, the irradiated groups showed improvement in all parameters in relation to the control group, indicating a retained capacity for angiogenesis after irradiation. PCR results confirmed the expression of osteogenesis-related genes in all irradiated groups. Dense collagen was detected 5 weeks after irradiation, and one construct showed discrete islands of bone indicating a retained osteogenic capacity after irradiation. This demonstrates for the first time that axial vascularization was capable of supporting a synthetic bone construct after a high dose of irradiation that is comparable to adjuvant radiotherapy. Copyright © 2016 John Wiley & Sons, Ltd.

Therapeutic options and postoperative wound complications after extremity soft tissue sarcoma resection and postoperative external beam radiotherapy.

Soft tissue sarcomas occur most commonly in the lower and upper extremities. The standard treatment is limb salvage surgery combined with radiotherapy. Postoperative radiotherapy is associated with wound complications. This systematic review aims to summarise the available evidence and review the literature of the last 10 years regarding postoperative wound complications in patients who had limb salvage surgical excision followed by direct closure vs flap coverage together with postoperative radiotherapy and to define the optimal timeframe for adjuvant radiotherapy after soft tissue sarcomas resection and flap reconstruction. A literature search was performed using PubMed. The following keywords were searched: limb salvage, limb-sparing, flaps, radiation therapy, radiation, irradiation, adjuvant radiotherapy, postoperative radiotherapy, radiation effects, wound healing, surgical wound infection, surgical wound dehiscence, wound healing, soft tissue sarcoma and neoplasms. In total, 1045 papers were retrieved. Thirty-seven articles were finally selected after screening of abstracts and applying dates and language filters and inclusion and exclusion criteria. Plastic surgery provides a vast number of reconstructive flap procedures that are directly linked to decreasing wound complications, especially with the expectant postoperative radiotherapy. This adjuvant radiotherapy is better administered in the first 3-6 weeks after reconstruction to allow timely wound healing and avoid local recurrence.

Pattern of Bone Generation after Irradiation in Vascularized Tissue Engineered Constructs.

BACKGROUND: Regenerative medicine modalities provide promising alternatives to conventional reconstruction techniques but are still deficient after malignant tumor excision or irradiation due to defective vascularization. METHODS: We investigated the pattern of bone formation in axially vascularized tissue engineering constructs (AVTECs) after irradiation in a study that mimics the clinical scenario after head and neck cancer. Heterotopic bone generation was induced in a subcutaneously implanted AVTEC in the thigh of six male New Zealand rabbits. The tissue construct was made up of Nanobone (Artoss GmbH; Rostock, Germany) granules mixed with autogenous bone marrow and 80 µL of bone morphogenic protein-2 at a concentration of 1.5 µg/µL. An arteriovenous loop was created microsurgically between the saphenous vessels and implanted in the core of the construct to induce axial vascularization. The constructs were subjected to external beam irradiation on postoperative day 20 with a single dose of 15 Gy. The constructs were removed 20 days after irradiation and subjected to histological and immunohistochemical analysis for vascularization, bone formation, apoptosis, and cellular proliferation. RESULTS: The vascularized constructs showed homogenous vascularization and bone formation both in their central and peripheral regions. Although vascularity, proliferation, and apoptosis were similar between central and peripheral regions of the constructs, significantly more bone was formed in the central regions of the constructs. CONCLUSION: The study shows for the first time the pattern of bone formation in AVTECs after irradiation using doses comparable to those applied after head and neck cancer. Axial vascularization probably enhances the osteoinductive properties in the central regions of AVTECs after irradiation.

The impact of various scaffold components on vascularized bone constructs.

Bone tissue engineering is gaining more interest in the field of craniofacial surgery where continuous efforts are being made to improve the outcomes via modulation of the scaffold components. In an in vitro three dimensional (3D) culture, the effect of bone morphogenic protein 2 (BMP2, 60 µg/ml) and the effect of different cell seeding densities (0.25, 0.5, and 1 × 104) of rat mesenchymal stem cells seeded on nanocrystalline hydroxyapatite in silica gel matrix (Nanobone®) on the cell viability and differentiation were studied. Alkaline phosphatase and viability assays were performed at day 7, day 14, and day 21 to assess the differentiation and the relative fraction of viable cells in the 3D cell cultures. In a subsequent in vivo study, we examined the effect of axial vascularization, the scaffold's particle size and the nature of the matrix (collagen type I vs. diluted fibrin) on vascularization and tissue generation in vascularized bone construct in rats. Regarding vascularization, we compared constructs vascularized randomly by extrinsic vascularization from the periphery of the implanted construct with others vascularized axially via an implanted arteriovenous loop (AVL). Regarding the particle size, we compared constructs having a scaffold particle size of 0.2 mm (powder) with other constructs having a particle size of 2 × 0.6 mm (granules). Regarding the matrix we compared constructs having a collagen matrix with others having a fibrin matrix. Various groups were compared regarding the amount of tissue generation, vascularization, and cellular proliferation. The initial seeding density had a temporary and minimal effect on the overall osteogenic differentiation of the cells. On the contrary, adding BMP2 in a concentration of 60 µg/ml over one week led to an overall enhanced osteogenic differentiation despite depressed cell viability. Axial vascularization was mandatory for efficient tissue formation and vascularization of the bone construct. Collagen matrix and a smaller particle size provided more favorable results in terms of vascularization and tissue formation than diluted fibrin and larger Nanobone particles.

Level I lymph node involvement in patients with N1b papillary thyroid carcinoma: a prospective study.

Most of the studies on the incidence, pattern, and predictive factors of lymph node (LN) metastasis with papillary thyroid carcinoma (PTC) have been performed retrospectively and no common consensus has been reached regarding the predictors for the involvement of level I LNs. This study was conducted prospectively to determine the incidence and the possible predictors of level I involvement in N1b PTC patients. The study included 30 consecutive patients with N1b stage of PTC. All the patients underwent neck dissection (ND) including level I. The relation between involvement of level I LNs and various clinicopathological variables was studied. Unilateral neck dissection was performed in 24 patients and bilateral neck dissection in six patients leading to 36 NDs. Level I was excised in all patients, with five specimens (14%) positive for metastasis. Levels II, III, IV, V, VI, and VII were positive in 52.8, 58.3, 58.3, 33.3, 63, and 22.2%, respectively. Level I involvement was significantly related to the number of lymph node levels affected (p = 0.003) and macroscopic extranodal invasion (p = 0.04). It was not related to the involvement of other individual levels, gender, age, size of the largest thyroid nodule, size of the largest LN involved, or histo-pathological variant of the tumor. This study suggests that including level I in therapeutic neck dissection for N1b PTC patients might be recommended in selected cases of multiple level involvement and macroscopic extranodal invasion requiring sacrifice of internal jugular vein, spinal accessory nerve, or sternomastoid muscle.

Axially vascularised mandibular constructs: Is it time for a clinical trial?

Applying regenerative therapies in the field of cranio-maxillofacial reconstruction has now become a daily practice. However, regeneration of challenging or irradiated bone defects following head and neck cancer is still far beyond clinical application. As the key factor for sound regeneration is the development of an adequate vascular supply for the construct, the current modalities using extrinsic vascularization are incapable of regenerating such complex defects. Our group has recently introduced the intrinsic axial vascularization technique to regenerate mandibular defects using the arteriovenous loop (AVL). The technique has shown promising results in terms of efficient vascularization and bone regeneration at the preclinical level. In this article, we have conducted a narrative literature review about using the AVL to vascularize tissue-engineering constructs at the preclinical level. We have also conducted a systematic literature review about applying the technique of axial vascularization in the field of craniofacial regeneration. The versatility of the technique and the possible challenges are discussed, and a suggested protocol for the first clinical trial applying the AVL technique for mandibular reconstruction is also presented.

Enhancing mandibular bone regeneration and perfusion via axial vascularization of scaffolds.

OBJECTIVE: Reconstruction of large and complex bone segments is a challenging problem facing maxillofacial surgery. The majority of current regenerative approaches rely on extrinsic vascularization, which is deficient after cancer ablation and irradiation. The aim of the study was to investigate the efficacy of intrinsic axial vascularization of synthetic bone scaffolds in the management of critical-size mandibular defects. MATERIALS AND METHODS: Scaffold-guided mandibular regeneration in two groups of adult male goats was compared. Only the scaffolds of the second group were axially vascularized via in situ embedding of an arteriovenous loop through microsurgical anastomosis of facial vessels. After 6 months of follow up, both groups were compared through radiological, biomechanical, histological and histomorphometric analysis. RESULTS: The axially vascularized constructs have showed significantly more central vascularization (p = 0.021) and markedly enhanced central bone formation (p = 0.08). The biomechanical characteristics were enhanced, but the difference between both groups was not statistically significant (p = 0.98). CONCLUSIONS: Axially vascularized synthetic mandibular grafts show better vascularization at their central regions, permitting more efficient bone regeneration. CLINICAL RELEVANCE: The encouraging results of the proposed technique could be of benefit in optimizing the reconstruction of large critical-size bone defects.

Salvage of a free radial forearm flap by creation of an arteriovenous fistula at the distal arterial pedicle.

Interdisciplinary approaches with infrainguinal bypass grafts and free flaps between the plastic and vascular surgery may well offer an opportunity for diabetic patients with peripheral vascular disease to salvage their critically affected limbs. A free flap transfer combined with an autologous vein graft can cover large tissue defects and simultaneously improve distal perfusion even in patients with arterial occlusive disease. We are presenting a case of bypass-free radial forearm flap used to cover a foot defect in an old diabetic patient with peripheral arterial disease. The flap perfusion deteriorated significantly during the early postoperative period. The patient was brought back to the operating room with acute thrombosis of the popliteal-radial venous graft and the arterial pedicle of the flap. The flap was salvaged by thrombectomy and creation of an additional arteriovenous fistula at the distal arterial pedicle. The procedure improved the flap perfusion and decreased the high internal resistance that was noticed in the flap when trying to flush the radial artery during the revision surgery and was evident by continuous wave -Doppler sonography. The successful salvage of the flap in the presented case and the convenient long-term follow up suggest that this technique may be safe and helpful as a last effort to salvage a bypass-free flap with a suspected high internal resistance.

Cultured keratinocytes on urinary bladder matrix scaffolds increase angiogenesis and help in rapid healing of wounds.

INTRODUCTION: Urinary Bladder Matrix (UBM) is an extracellular matrix (ECM) scaffold. It is now used in wound care management of partial and full-thickness wounds where conventional methods for wound care usually fail to give satisfactory results. OBJECTIVE: In this study, the authors are comparing the healing of full-thickness excisional wounds in New Zealand rabbits using either UBM scaffolds alone or in combination with cultured keratinocytes. The wounds were compared grossly and histologically. MATERIALS AND METHODS: It is a comparative controlled study including 40 full-thickness wounds in 2 groups. Group (A) wounds: treated with UBM scaffolds, Group (B) wounds: treated with UBM scaffolds with cultured keratinocytes. The wounds were examined grossly after 1, 2, and 3 weeks, and were examined histologically at the end of the 3rd week using ordinary hematoxylin-eosin staining techniques. RESULTS: All the wounds healed completely by the end of the 3rd week. Early wound contraction was significantly less in group B. More angiogenic response was evident in all specimens of group B. CONCLUSION: This study shows that adding cultured keratinocytes to the rough surface of the UBM scaffold may be beneficial in reducing early wound contraction and improving wound vascularity in treatment of full-thickness wounds.

Mandibular reconstruction using an axially vascularized tissue-engineered construct.

BACKGROUND: Current reconstructive techniques for continuity defects of the mandible include the use of free flaps, bone grafts, and alloplastic materials. New methods of regenerative medicine designed to restore tissues depend mainly on the so-called extrinsic neovascularization, where the neovascular bed originates from the periphery of the construct. This method is not applicable for large defects in irradiated fields. METHODS: We are introducing a new animal model for mandibular reconstruction using intrinsic axial vascularization by the Arterio-Venous (AV) loop. In order to test this model, we made cadaveric, mechanical loading, and surgical pilot studies on adult male goats. The cadaveric study aimed at defining the best vascular axis to be used in creating the AV loop in the mandibular region. Mechanical loading studies (3 points bending test) were done to ensure that the mechanical properties of the mandible were significantly affected by the designed defect, and to put a base line for further mechanical testing after bone regeneration. A pilot surgical study was done to ensure smooth operative and post operative procedures. RESULTS: The best vascular axis to reconstruct defects in the posterior half of the mandible is the facial artery (average length 32.5 ± 1.9 mm, caliber 2.5 mm), and facial vein (average length 33.3 ± 1.8 mm, caliber 2.6 mm). Defects in the anterior half require an additional venous graft. The defect was shown to be significantly affecting the mechanical properties of the mandible (P value 0.0204). The animal was able to feed on soft diet from the 3rd postoperative day and returned to normal diet within a week. The mandible did not break during the period of follow up (2 months). CONCLUSIONS: Our model introduces the concept of axial vascularization of mandibular constructs. This model can be used to assess bone regeneration for large bony defects in irradiated fields. This is the first study to introduce the concept of axial vascularization using the AV loop for angiogenesis in the mandibular region. Moreover, this is the first study aiming at axial vascularization of synthetic tissue engineering constructs at the site of the defect without any need for tissue transfer (in contrast to what was done previously in prefabricated flaps).