Freeze-Dried Mesenchymal Stem Cells: From Bench to Bedside. Review.

This review describes the freeze-dried mesenchymal stem cells (MSCs) and their ability to restore damaged tissues and organs. An analysis of the literature shows that after the lyophilization MSCs retain >80% of paracrine factors and that the mechanism of their action on the restoration of damaged tissues and organs is similar to the mechanism of action of paracrine factors in fresh and cryopreserved mesenchymal stem cells. Based on the own materials, the use of paracrine factors of freeze-dried MSCs in vivo and in vitro for the treatment of various diseases of organs and tissues has shown to be effective. The study also discusses about the advantages and disadvantages of freeze-dried MSCs versus cryopreserved MSCs. However, for the effective use of freeze-dried MSCs in clinical practice, a more detailed study of the mechanism of interaction of paracrine factors of freeze-dried MSCs with target cells and tissues is required. It is also necessary to identify possible other specific paracrine factors of freeze-dried MSCs. In addition, develop new therapeutic strategies for the use of freeze-dried MSCs in regenerative medicine and tissue bioengineering.

Intravital tumor decellularization as a new approach to cancer treatment.

This study demonstrates the possibility of tumor decellularization in living animals. Subcutaneous Ehrlich tumor induced by isolated Ehrlich ascitic carcinoma cells in mice was used as a model. The study also presents methods for ex vivo decellularization of human gastric adenocarcinoma (HGA) and hepatocellular carcinoma (HCC) induced by diethylnitrosamine (DEN) in rat. Sodium dodecyl sulfate (SDS) and Triton X-100 were used as detergents for tumor decellularization. The detergents for HGA and HCC were administered through organ vessels. For intravital decellularization of Ehrlich's subcutaneous tumor, detergents were injected directly into the tumor parenchyma. The results of the study showed that the effectiveness of tumor decellularization using SDS and Triton X-100 depended on the size, structure, stiffness and density of the tumor, as well as on the concentration, route and speed of detergent administration. The study also showed that an hour after the initiation of decellularization, the central part of Ehrlich's tumor changed the color, and after three hours, it completely acquired a translucent white color. Chemical contamination of tissues surrounding the tumor with the detergents was not observed. Histological studies showed the complete absence of all cellular components of Ehrlich's tumor and a slightly deformed extracellular matrix (ECM). There were no loco-regional recurrences or metastases of Ehrlich's tumor within 150 days after decellularization. The developed intravital decellularization method allows the effective removal of the cellular components and the DNA content of Ehrlich's subcutaneous tumor without compromising animal health. Additionally, this method can destroy tumor ECM, which will significantly improve the delivery of anticancer drugs to the tumor cells. However, more detailed and extensive studies are needed to develop an in vivo technique for isolated decellularization of the tumor or a part of the organ with the tumor. It is also necessary to identify less toxic decellularization agents and to develop the most efficient route for their delivery to the tumor cells.

Challenges and Opportunities Associated With Drug Delivery for the Treatment of Solid Tumors.

In this review, we discuss the effectiveness of drug delivery system based on metal nanoparticles, and also, describe the problems associated with their delivery to tumor cells. Throughout recent years, more reports have appeared in the literature that demonstrate promising results for the treatment of various types of cancer using metal-based nanoparticles. Due to their unique physical and chemical properties, metal nanoparticles are effectively being used for the delivery of drug to the tumor cells, for cancer diagnosis and treatment. They can also be synthesized allowing the control of size and shape. However, the effectiveness of the metal nanoparticles for cancer treatment largely depends on their stability, biocompatibility, and ability to selectively affect tumor cells after their systemic or local administration. Another major problem associated with metal nanoparticles is their ability to overcome tumor tissue barriers such as atypical blood vessel structure, dense and rigid extracellular matrix, and high pressure of tumor interstitial fluid. The review also describes the design of tumor drug delivery systems that are based on metal nanoparticles. The mechanism of action of metal nanoparticles on cancer cells is also discussed. Considering the therapeutic safety and toxicity of metal nanoparticles, the prospects for their use for future clinical applications are being currently reviewed.

A novel therapeutic vaccine targeting the soluble TNFα receptor II to limit the progression of cardiovascular disease: AtheroVax™.

The burden of atherosclerotic cardiovascular disease contributes to a large proportion of morbidity and mortality, globally. Vaccination against atherosclerosis has been proposed for over 20 years targeting different mediators of atherothrombosis; however, these have not been adequately evaluated in human clinical trials to assess safety and efficacy. Inflammation is a driver of atherosclerosis, but inflammatory mediators are essential components of the immune response. Only pathogenic forms of sTNFR2 are acted upon while preserving the membrane-bound (wild-type) TNFR2 contributions to a non-pathogenic immune response. We hypothesize that the inhibition of sTNRF2 will be more specific and offer long-term treatment options. Here we describe pre-clinical findings of an sTNFR2-targeting peptide vaccine (AtheroVax™) in a mouse model. The multiple pathways to synthesis of the soluble TNFRII receptor (sTNFRII) were identified as sTNFRII(PC), sTNFRII(Δ7), and sTNFRII(Δ7,9). The sTNFRII(Δ7) peptide, NH2-DFALPVEKPLCLQR-COOH is specific to sTNFR2 based on an mRNA splice-variant in which exon 6 is joined to exon 8. The role of sTNFRII(Δ7) as a mediator of prolonged TNFα activity by preventing degradation and clearance was investigated. Inflammation is a critical driver of onset, progression and expansion of atherosclerosis. The TNFα ligand represents a driver of inflammation that is mediated by a splice variant of TNFR2, referred to as sTNFRII(Δ7). The multiple forms of TNFRII, both membrane bound and soluble, are associated with distinctly different phenotypes. sTNFRII(PC) and sTNFRII(Δ7) are not equivalent to etanercept because they lack a clearance mechanism. The unique peptide associated with sTNFRII(Δ7) contains a linear B-cell epitope with amino acids from both exon 6 and exon 8 supporting the vaccine design. Animal studies to evaluate the vaccine are ongoing, and results will be forthcoming. We describe a peptide vaccine targeting sTNFR2 in limiting the progression of atherosclerosis. A therapeutic vaccine limiting the progression of atherosclerosis will greatly contribute to the reduction in morbidity and mortality from cardiovascular disease. It is likely the vaccine will be used in combination with the current standards of care and lifestyle modifications.

Multifunctional hybrid hydrogel for the prevention of post-surgery tumor recurrence.

In this study, we present a multifunctional hybrid hydrogel (MFHH) for the prevention of postoperative tumor recurrence. MFHH consists of two components; component A - containing a gelatin-based cisplatin, which destroys the residual cancer after surgery, and component B - containing macroporous gelatin microcarriers (CultiSpher) loaded with freeze-dried bone marrow stem cells (BMSCs), which activates the wound healing process. We also evaluated the effects of MFHH in a subcutaneous Ehrlich tumor mouse model. MFHH acted as a local delivery system by directly supplying cisplatin to the tumor environment, resulting in excellent anti-cancer effects and minimal side effects. MFHH released cisplatin gradually to destroy the residual tumors, thereby preventing loco-regional recurrence. We have also demonstrated that BMSCs are able to inhibit residual tumor growth. Moreover, CultiSpher loaded with BMSCs acted as an injection 3D scaffold and easily filled the wound defect formed by tumor removal, and the paracrine factors of the freeze-dried BMSCs accelerated the wound healing process. The components of the MFHH can be used both separately and together. However, for the successful application of MFHH in clinical practice, it is necessary to study in more detail the role of paracrine factors of freeze-dried BMSCs in the inhibition or proliferation of residual cancer. These questions will be the focus of our future research.

Local drug delivery system for the treatment of tongue squamous cell carcinoma in rats.

The present study describes a local drug delivery system with two functions, which can suppress tumor growth and accelerate wound healing. Thе system consists of a two-layer multicomponent fibrin-based gel (MCPFTG). The internal layer of MCPFTG, which is in direct contact with the wound surface, contains cisplatin placed on a CultiSpher-S collagen microcarrier. The external layer of MCPFTG consists of a CultiSpher-S microcarrier with lyophilized bone marrow stem cells (BMSCs). The efficacy of MCPFTG was evaluated in a rat model of squamous cell carcinoma of the tongue created with 4-nitroquinoline 1-oxide. The results of the study showed that, within 20-25 days, a non-healing wound of the tongue was formed in animals that underwent only 85% resection of squamous cell carcinoma, while rapid progression of the residual tumor was concomitantly observed. Immunohistochemical methods revealed high expression of cyclin D1 and low expression of E-cadherin in these animals. Additionally, high expression of p63 and Ki-67 was noted. In 80% of animals with squamous cell carcinoma of the tongue that were treated with MCPFTG after 85% tumor resection, a noticeable suppression of tumor growth was evident throughout 150 days, and tumor recurrence was not detected. Immunohistochemistry revealed low or moderate expression of cyclin D1, and high expression of E-cadherin throughout the whole observation period. The MCPFTG-based local drug delivery system was shown to be effective in suppressing tumor growth and preventing recurrence. MCPFTG decreased the toxicity of cisplatin and enhanced its antitumor activity. In addition, lyophilized paracrine BMSC factors present in MCPFTG accelerated wound healing after tumor removal. Thus, the present study suggests novel opportunities for the development of a multifunctional drug delivery system for the treatment of squamous cell carcinoma.

Challenges and perspectives of surgical treatment of liver failure. Current status and last achievements in Georgia.

Liver transplantation is considered to be the last hope of treatment for irreversible liver failure caused by different diffuse and/or space-occupying lesions of this organ. The strict limitation of the donor organs stipulates for development of alternative approaches for the solving this problem. The presented review of literature and our experience aims to discuss the modern aspects of management of different hepatic pathologies causing liver failure with the view of creation of the auxiliary, bioengineer-based functional tissues and/or organs and innovative surgical interventions allowing to conduct the operations in cases, which were up to date considered as inoperable. There are highlighted the last achievements of the experimental and translational studies performed in four University research centers of Georgia, which, on the one hand, provoke the specific professional interest, and on the other hand, require the international cooperation and collaboration for further progress and advances in this field of surgery. KEY WORDS: Artificial liver, Bio-Artificial organs, Liver failure, Innovative surgery, Tissue engineering.

Oral microbiota and oral cancer: Review.

In this review, we draw attention and discuss the risk factors and causes of the development of oral squamous cell carcinoma (OSCC) focusing on oral microbiota. Recently, a breakthrough in the study of cancer has been the discovery of the relationship between the presence of certain types of bacteria and the development of cancer in the human body. Studies have shown that, Porphyromonas gingivalis (P. gingivalis) bacteria that is responsible for the destructive processes in the oral cavity, could play an important role in the development of OSCC. In our continuing search for bacteria that causes oral squamous cell carcinoma, we came across the Pseudomona aeruginosa, which due to its metabolite properties, may play important role in carcinogenesis of oral cancer. One possible mechanism is the ability of Pseudomonas to synthesize nitric oxide (NO) that modulates different cancer-related appearances such as apoptosis, cell cycle, angiogenesis, invasion, and metastasis. We think that P. aeruginosa increases the concentration of NO by converting salivary nitrite to nitric oxide, and this is how it contributes to NO-related carcinogenesis. Early diagnosis and treatment of periodontitis are very important not only for patients' oral health, but also for the prevention of OSCC development. Screening test for OSCC based on determination of salivary NO levels could be appealing and may prove to be useful assay for diagnosis and early detection of disease progression in oral cancer.

Bone Marrow Stem Cell and Decellularized Human Amniotic Membrane for the Treatment of Nonhealing Wound After Radiation Therapy.

OBJECTIVES: In this study, our aim was to create a bioactive wound dressing that combined decellularized and lyophilized human amniotic membrane and freeze-dried rat bone marrow stem cells for the treatment of nonhealing wounds. MATERIALS AND METHODS: For the decellularized human amniotic membrane, sodium dodecyl sulfate and 1% Triton X-100 were used. The mononuclear fraction of bone marrow stem cells was isolated by density gradient centrifugation using Ficoll Paque Plus (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Investigations were conducted on Lewis inbred rats with the radiation wound model (dose of 60 Gy). On day 20 after application of radiation, the skin was excised around the radiation burn. The wound was treated with decellularized human amniotic membrane seeded with and without freeze-dried bone marrow stem cells. RESULTS: The use of a decellularized amniotic membrane for closing the burn wound increased the rate of healing by 2.5 times; the use of a decellularized amniotic membrane seeded with bone marrow stem cells or freeze-dried bone marrow stem cells increased the rate of wound healing by approximately 4 times. CONCLUSIONS: Administration of freeze-dried bone marrow stem cell may represent a novel therapeutic approach in the treatment of nonhealing wounds and other conditions. We observed no evidence of local or systemic complications related to the procedure. However, further efforts with better protocol design for future studies are needed.

A three-dimensional scaffold from decellularized human umbilical artery for bile duct reconstruction.

AIM: The main purpose of the study was to create in vitro bile duct equivalent out of decellularized human umbilical cord artery and use it to reconstruct common bile duct obstruction with preservation of sphincter of Oddi. MATERIAL AND METHODS: SDS and Triton X-100 were used for decellularization of the artery. Allogeneic isolated cholangiocytes were seeded onto the inner surface of the decellularized artery. Experimental study was held and 12 domestic pigs of both sexes, weighing 25-30 kg were used. They were divided in equivalent two groups. Common bile duct obstruction model was created in all animals. Animals of the first group (n=6) received no further treatment and were under observation. Animals of the second group (n=6) underwent relaparotomy after two days of initial intervention, lesion site (2 cm) was incised and defect was reconstructed with the bile duct equivalent with the size of 2-2,5 cm. Maximum observation period was 84 days. RESULTS: Laboratory, morphologic and radiologic investigations showed good integration with the host organism. DISCUSSION: Bile duct reconstruction is still a major of HPB surgery. This fact prompted this study to assess the efficacy of the novel method for bile duct reconstruction the experimental study by using appropriate laboratory, morphologic and radiologic investigations. CONCLUSION: Preliminary results obtained with the described method allows us to say that bile duct equivalent created by us with decellularized human umbilical artery and cholangiocytes can be successfully used for bile duct reconstruction with inclusion of the sphincter of oddi. KEY WORDS: Bile Duct Obstruction, Bile Duct Reconstruction, Decellularization, Human Umbilical Artery.

Reconstruction of mandibular defects with autogenous bone and decellularized bovine bone grafts with freeze-dried bone marrow stem cell paracrine factors.

The gold standard following segmental mandibulectomy is vascularized autologous bone graft in the form of the fibula flap. However, in bone reconstruction the use of autogenous bone does not always guarantee a successful outcome. The aim of the present investigation was to develop a novel biologically active bone (BAB) graft, and to use it for the reconstruction of large size defects of the mandible bone following tumor resection. In the first part of the present study, biologically active bone graft was developed by using human freeze-dried bone marrow stem cells (BMSCs) paracrine factors and three-dimensional bone scaffold derived from cancellous bovine bone following decellularization. In the second part of the research, one male and three female patients with primary tumors of the mandible underwent hemimandibulectomy. The mandibular bone defects following tumor resection were reconstructed with autogenous rib grafts in three patients and BAB graft was used in one patient. The graft-host interfaces were covered with decellularized human amnion/chorion membrane graft. All patients were followed-up every five months following the reconstruction of the mandible, with no complications observed. Preliminary clinical investigations demonstrated that a BAB graft containing freeze-dried BMSC paracrine factors may be used for the reconstruction of large mandibular bone defects following tumor resection.

A Novel Endoscopic Bariatric Procedure: Results of an Experimental Study.

BACKGROUND: Surgical treatment of obesity is characterized by both early and late complications, and thus, there is a need to develop safe and non-invasive techniques. Ghrelin is an orexigenic hormone produced by the fundus of the stomach, which may represent a novel target for obesity management. Unfortunately, numerous attempts to alter ghrelin levels have failed to present significant clinical results. We describe a novel procedure that involves modifying arterial blood flow to the gastric fundus for limiting plasma ghrelin levels. METHODS: A gastroscope was advanced into the gastric fundus of 13 healthy Yorkshire swine, and the fundus was clipped under direct visualization to restrict left gastric artery blood flow. Body weights and ghrelin levels were recorded before and once a week for 4 weeks after the procedure. RESULTS: Compared to controls, gastroscopic clipping of the fundus decreased plasma ghrelin levels and prevented further weight gain in the 4 weeks of follow-up. Immunohistochemistry and histomorphometry revealed reduced numbers of ghrelin-positive cells in the fundus of experimental animals. We also observed thrombosis in submucosal arteries and submucosal fibrosis. Histological studies demonstrated minimal gastric mucosal injury. CONCLUSION: Gastroscopic clipping of the fundus in an experimental porcine model resulted in sustained weight loss and a reduction in plasma ghrelin levels at 1 month post-procedure, with no adverse events. Further experimental studies in human patients are needed to examine the clinical utility of this procedure and to optimize a technique, which can facilitate adequate weight loss while minimizing the risk of mucosal injury.

Phase 1 Trial of Autologous Bone Marrow Stem Cell Transplantation in Patients with Spinal Cord Injury.

Introduction. A total of 18 patients, with complete motor deficits and paraplegia caused by thoracic and lumbar spine trauma without muscle atrophy or psychiatric problems, were included into this study. Materials and Methods. The bone marrow was aspirated from the anterior iliac crest under local anesthesia and the mononuclear fraction was isolated by density gradient method. At least 750 million mononuclear-enriched cells, suspended in 2 mL of saline, were infused intrathecally. Results and Discussion. The study reports demonstrated improvement of motor and sensory functions of various degrees observed in 9 of the 18 (50%) cases after bone marrow stem cell transplantation. Measured by the American Spinal Injury Association (ASIA) scale, 7 (78%) out of the 9 patients observed an improvement by one grade, while two cases (22%) saw an improvement by two grades. However, there were no cases in which the condition was improved by three grades. Conclusions. Analysis of subsequent treatment results indicated that the transplantation of mononuclear-enriched autologous BMSCs is a feasible and safe technique. However, successful application of the BMSCs in the clinical practice is associated with the necessity of executing more detailed examinations to evaluate the effect of BMSCs on the patients with spinal cord injury.

Clinical application of decellularized and lyophilized human amnion/chorion membrane grafts for closing post-laryngectomy pharyngocutaneous fistulas.

BACKGROUND AND OBJECTIVES: Squamous cell carcinoma is the most common pathological type among the cancers of the larynx. Standard treatment for squamous cell carcinoma of the larynx is the combination of chemotherapy, radiotherapy, and laryngectomy. Pharyngocutaneous fistula is a common complication of laryngectomy. We hypothesized that decellularized and lyophilized human amnion/chorion membrane can be an effective, non-invasive method of treating pharyngocutaneous fistula. METHODS: A total of 67 patients with laryngeal squamous cell carcinoma were retrospectively analyzed after treatment in a prospective trial. After preoperative chemotherapy, radiotherapy, and total or extended laryngectomy, primary wound healing occurred in 42 (62.7%) patients. Pharyngocutaneous fistula developed in 8 (11.9%) patients. Decellularized and lyophilized human amnion/chorion membrane grafts were used to reconstruct the fistulas. RESULTS: The average time for the full healing of the wound in all patients after transplantation of these grafts was 18 days. CONCLUSION: The advantages of using these grafts over other existing methods of pharyngocutaneous fistula treatment are that they are non-invasive, prevent donor morbidity, and enable management of the wound without using classical wound gauze. J. Surg. Oncol. 2016;113:538-543. © 2016 Wiley Periodicals, Inc.

Benign osteoblastoma of the mandible in a 12-year-old female: A case report.

Benign osteoblastoma refers to a benign tumor of the bone. Osteoblastoma most commonly affects the vertebrae and long tubular bones, however, in rare cases is observed in the facial bones. The current study presents the case of a 12-year-old female patient with a tumor in the mandibular body. Radiological imaging revealed a lesion with regular contours. The lesion was radically resected and histological analysis of the specimen demonstrated features that are typical of a benign osteoblastoma. The consequential defects of the jaw were reconstructed using titanium implants and autologous bone transplantation. The patient remains disease free subsequent to a five-month follow-up period. The aim of the present report is to present a rare case of benign osteoblastoma of the mandible. This study demonstrated that correct diagnosis and complete surgical excision are important to reduce the risk of recurrence of a benign osteoblastoma.

Petrous carotid artery's in situ bypass: anatomic study.

The purpose of our study was to reveal the possibility of cervical-to-petrous carotid artery in situ bypass with maximum preservation of cranial nerves. Four human cadavers prepared in formalin were investigated. Eight surgical approaches were used (bilateral exposure on each cadaver). The skin incision started from the level of the temporomandibular joint. The VII, IX, X, and XII cranial nerves, starting from the stylomastoid angle, were maximally preserved. Resection of the styloid process, subluxation of the mandibular joint, and milling of tympanic bone revealed the petrous carotid artery. Exposure of the carotid bifurcation was performed with a mini-skin incision. After dividing the petrous internal carotid artery (ICA) and the cervical ICA, the cervical ICA was dilated using a Fogarty catheter to simulate aneurysmal dilatation. The patient's saphenous vein after stripping was utilized for the bypass. The vein was passed into the lumen of the dilated cervical ICA, and a cervical-to-petrous carotid bypass was performed. In each case, the described technique made it possible to expose the intrapetrous carotid artery adequately. In two cases it was impossible to make a luxation, and therefore the mandibular branch was resected. The vertical segment's mean length was 12 mm (range 10-15 mm). In all cases, the VII, IX, X, and XII cranial nerves were preserved maximally. The main points of the approach are luxation of the mandibular articulation and milling of the tympanic bone. Our study in cadavers suggests the possibility of petrous carotid artery bypass without exposing the cervical ICA.

Isolated small intestinal segments support auxiliary livers with maintenance of hepatic functions.

We determine here the functional integrity of auxiliary livers in containers fashioned from the small intestine. Liver microfragments from dipeptidyl peptidase 4 (DPP4)-deficient rats were transplanted into syngeneic normal animals with isolated intestinal segments characterized by mucosal denudation but intact vascular supply. Transplanted liver fragments were restored to confluent tissue with normal hepatic architecture and development of DPP4-positive vessels, indicating angiogenesis and revascularization. Auxiliary liver units expressed multiple hepatotrophic and angiogenic genes, and transplanted tissues remained intact for up to the 6-week duration of the studies with neither ischemic injury nor significant hepatocellular proliferation. Hepatic metabolic, transport and synthetic functions were preserved in auxiliary livers, including uptake and biliary excretion of (99m)Tc-mebrofenin in syngeneic recipients of liver from F344 rats, as well as secretion of albumin in allografted Nagase analbuminemic rats. This ability to produce functionally competent auxiliary livers in vascularized intestinal segments offers therapeutic potential for liver disease and genetic deficiency.