Surface characteristics of biomaterials used for space maintenance in a mandibular defect: a pilot animal study.

PURPOSE: The purpose of the present study was to evaluate the effect of implant porosity on wound healing between solid and porous implants placed within a bony mandibular defect with intraoral exposure. MATERIALS AND METHODS: Solid poly(methyl methacrylate) (PMMA) implants similar to those used currently in clinical space maintenance applications in maxillofacial surgery were compared with poly(propylene fumarate) implants that contained a porous outer surface surrounding a solid core. A 10-mm diameter nonhealing bicortical defect with open communication into the oral cavity was created in the molar mandibular region of 12 adult male New Zealand white rabbits. Of the 12 rabbits, 6 received the hybrid poly(propylene fumarate) implants and 6 received the solid PMMA implants. At 12 weeks, the rabbit mandibles were harvested and sent for histologic staining and sectioning. RESULTS: Gross inspection and histologic examination showed all 6 poly(propylene fumarate) implants to be intact within the defect site at the termination of the study period, with 3 of the 6 specimens exhibiting a continuous circumferential soft tissue margin. In contrast, 5 of the 6 PMMA-implanted specimens were exposed intraorally with an incomplete cuff of soft tissue around the implant. One of the PMMA-implanted specimens exhibited complete extrusion and subsequent loss of the implant. Fisher's exact test was used to compare the occurrence of oral cavity wound healing between the 2 groups (P = .09). CONCLUSIONS: Although statistically significant differences between the 2 groups were not seen, our results have indicated that advantages might exist to using porous implants for space maintenance. Additional study is needed to evaluate these findings.

Smartwatch inertial sensors continuously monitor real-world motor fluctuations in Parkinson's disease.

Longitudinal, remote monitoring of motor symptoms in Parkinson's disease (PD) could enable more precise treatment decisions. We developed the Motor fluctuations Monitor for Parkinson's Disease (MM4PD), an ambulatory monitoring system that used smartwatch inertial sensors to continuously track fluctuations in resting tremor and dyskinesia. We designed and validated MM4PD in 343 participants with PD, including a longitudinal study of up to 6 months in a 225-subject cohort. MM4PD measurements correlated to clinical evaluations of tremor severity (ρ = 0.80) and mapped to expert ratings of dyskinesia presence (P < 0.001) during in-clinic tasks. MM4PD captured symptom changes in response to treatment that matched the clinician's expectations in 94% of evaluated subjects. In the remaining 6% of cases, symptom data from MM4PD identified opportunities to make improvements in pharmacologic strategy. These results demonstrate the promise of MM4PD as a tool to support patient-clinician communication, medication titration, and clinical trial design.

Synthesis and characterization of dual stimuli responsive macromers based on poly(N-isopropylacrylamide) and poly(vinylphosphonic acid).

Stimulus responsive materials hold great promise in biological applications as they can react to changes in physiological stimuli to produce a desired effect. Stimulus responsive macromers designed to respond to temperature changes at or around 37 degrees C and the presence of divalent cations were synthesized from N-isopropylacrylamide, pentaerythritol diacrylate monostearate, 2-hydroxyethyl acrylate, and vinylphosphonic acid by free radical polymerization. Monomers were incorporated into the macromers in ratios approximating the molar feed ratios, and macromers underwent thermogelation around normal body temperature (36.2-40.5 degrees C) as determined by rheology and differential scanning calorimetry. Macromers containing vinylphosphonic acid interacted with calcium ions in solution, displaying decreased sol-gel transition temperatures (27.6-34.4 degrees C in 100 mM CaCl(2)), with decreases of greater magnitude observed for macromers with higher relative vinylphosphonic acid content. Critical micellar concentrations also decreased in a dose-dependent manner with increased vinylphosphonic acid incorporation in solutions with CaCl(2) but not in solutions with NaCl. These dually responsive macromers allow examination of the effect of increasing vinylphosphonic acid content in a macromer, which holds promise in biological applications such as drug and cell delivery or tissue engineering due to the macromer responsiveness at physiological temperatures and concentrations of calcium.

Evaluation of lymphatic function by means of dynamic Gd-BOPTA-enhanced MRL in experimental rabbit limb lymphedema.

BACKGROUND: The aim of this study was to investigate the value and technical methods of 3D dynamic contrast-enhanced magnetic resonance lymphangiography (MRL) in the assessment of lymphatic anatomy and function in the presence of extremity lymphedema. MATERIAL/METHODS: An improved experimental model of obstructive lymphedema was established in 1 hind limb of 6 New Zealand White rabbits. 3D contrast-enhanced MRL was performed with a 3.0-T MR unit after the intracutaneous injection of Gd-BOPTA into the interdigital webs of the dorsal paws. Maximum-intensity projection (MIP) was used to reconstruct the images of the lymphatic system. The dynamic nodal enhancement in the popliteal fossa and time-signal intensity curves between lymphedematous and contralateral limbs were compared. Morphologic abnormalities of the lymphatic system were also evaluated and compared with lymphoscintigraphy (LSG). RESULTS: 3D dynamic contrast-enhanced MRL images were obtained after the administration of Gd-BOPTA. In the normal limb, the popliteal fossa lymph nodes and their afferent and efferent lymph-collecting vessels were clearly visualized as the Gd tracer was rapidly cleared from the interstitial compartment. In contrast, the Gd tracer accumulated slowly at the prior surgical site in the lymphedematous limb. The nodal enhancement of lymphedematous limbs was significantly less than that of the contralateral limbs (P<0.01). Types of time-signal intensity curves were also significantly different between the 2 groups (P<0.001). CONCLUSIONS: 3D dynamic contrast-enhanced MRL can visualize the precise anatomy of lymphatic vessels and lymph nodes in extremity lymphedema, as well as objectively evaluate the functional status of lymph flow transport.

Harvesting and cryopreservation of lymphatic endothelial cells for lymphatic tissue engineering.

In order to provide a suitable source of cells for lymphatic tissue engineering, the present study was designed to investigate techniques for harvesting and cryopreservation of human dermal lymphatic endothelial cells (LECs) in vitro. The LECs were isolated from children's foreskins and then cultured in endothelial growth medium-2 MV (EGM-2-MV) with 5% FBS. The second passage LECs were suspended in cryopreservation solution containing 40% FBS and 10% Me(2)SO in EGM-2-MV, cooled to -80 degrees C at about 1 degrees C/min and stored in liquid nitrogen. Samples were thawed quickly in a 37 degrees C water bath, and the cryoprotectant was removed by serial elution. The membrane integrity of thawed LECs was determined by trypan blue staining exclusion, and their proliferation was evaluated using the MTT method. The expanded cells of two groups were identified using immunofluorescence staining and RT-PCR with lymphatic-specific markers such as Podoplanin and VEGFR-3. Uptake of fluorescent DiI-Ac-LDL and microtubular formation in three-dimensional cultures were used to detect the function of LECs. Flow cytometry was applied to identify cells and to measure the apoptosis rate as well. Cryopreservation resulted in a retrieval of 67+/-4% and an intact cell rate of 80+/-3%. The early apoptosis rate of thawed LECs (9.15+/-0.34%) was higher than that of fresh control LECs (5.31+/-0.23%). The growth curves of thawed LECs were similar to those of fresh LECs. The thawed LECs were propagated for at least 6-7 passages without alterations in phenotype and function. Highly purified LECs can be isolated by immunomagnetic beads from human dermis. The cryopreserved/thawed and recultivated LECs are proven to have high vitality and growth potential in vitro and may be considered suitable seed cells for lymphatic tissue engineering.

Adenoviral transduction of hTGF-beta1 enhances the chondrogenesis of bone marrow derived stromal cells.

TGF-beta1 plays a necessary and important role in the induction of chondrogenic differentiation of bone marrow stromal cells (BMSCs). In this study, porcine BMSCs were infected with a replication-deficient adenovirus expression vector carrying the hTGF-beta1 gene. The transduced BMSCs were cultured as pelleted micromasses in vitro for 21 days, seeded onto disk-shaped PGA scaffolds for 3 days and subsequently implanted into the subcutaneous tissue of mice. BMSCs transduced with AdhTGF-beta1 expressed and secreted more hTGF-beta1 protein in vitro than those of the control group. Histological and immunohistological examination of the pellets revealed robust chondrogenic differentiation. Tissues made from cells transduced with AdhTGF-beta1 exhibited neocartilage formation after 3 weeks in vivo. The neocartilage occupied 42 +/- 5% of the total tissue volume which was significantly greater than that of the control group. Furthermore, there was extensive staining for sulfated proteoglycans and type II collagen in the AdhTGF-beta1 group compared to controls, and quantification of GAG content showed significantly greater amounts of GAG in experimental groups. The results demonstrate that transfer of hTGF-beta1 into BMSCs via adenoviral transduction can induce chondrogenic differentiation in vitro and enhance chondrogenesis in vivo.

Purification of Schwann cells from adult rats by differential detachment.

Differential detachment by collagenase treatment is a new and efficient method for Schwann cell (SC) purification. As its effect on adult animals remains unclear, we have investigated the possibility of SC purification from adult rats. To avoid any systematic bias, Schwann cell purity before and after purification were compared by morphology, immunostaining of P75(NTR) and S100 and flow cytometric analysis. The final SC purities reached 99% as confirmed by three independent analyses SC purity and the cell yields were above 10(6) cells after two rounds of purification. The method of differential detachment is also suitable for SC purification in adult rats and could be useful for research and clinical applications.

Donor age and cell passage affects differentiation potential of murine bone marrow-derived stem cells.

BACKGROUND: Bone marrow-derived mesenchymal stem cells (BMSCs) are a widely researched adult stem cell population capable of differentiation into various lineages. Because many promising applications of tissue engineering require cell expansion following harvest and involve the treatment of diseases and conditions found in an aging population, the effect of donor age and ex vivo handling must be understood in order to develop clinical techniques and therapeutics based on these cells. Furthermore, there currently exists little understanding as to how these two factors may be influenced by one another. RESULTS: Differences in the adipogenic, chondrogenic, and osteogenic differentiation capacity of murine MSCs harvested from donor animals of different age and number of passages of these cells were observed. Cells from younger donors adhered to tissue culture polystyrene better and proliferated in greater number than those from older animals. Chondrogenic and osteogenic potential decreased with age for each group, and adipogenic differentiation decreased only in cells from the oldest donors. Significant decreases in differentiation potentials due to passage were observed as well for osteogenesis of BMSCs from the youngest donors and chondrogenesis of the cells from the oldest donors. CONCLUSION: Both increasing age and the number of passages have lineage dependent effects on BMSC differentiation potential. Furthermore, there is an obvious interplay between donor age and cell passage that in the future must be accounted for when developing cell-based therapies for clinical use.

Synthesis and characterization of injectable, thermally and chemically gelable, amphiphilic poly(N-isopropylacrylamide)-based macromers.

In this study, we synthesized and characterized a series of macromers based on poly( N-isopropylacrylamide) that undergo thermally induced physical gelation and, following chemical modification, can be chemically cross-linked. Macromers with number average molecular weights typically ranging from 2000-3500 Da were synthesized via free radical polymerization from, in addition to N-isopropylacrylamide, pentaerythritol diacrylate monostearate, a bifunctional monomer containing a long hydrophobic chain, acrylamide, a hydrophilic monomer, and hydroxyethyl acrylate, a hydrophilic monomer used to provide hydroxyl groups for further chemical modification. Results indicated that the hydrophobic-hydrophilic balance achieved by varying the relative concentrations of comonomers used during synthesis was an important parameter in controlling the transition temperature of the macromers in solution and stability of the resultant gels. Storage moduli of the macromers increased over 4 orders of magnitude once gelation occurred above the transition temperature. Furthermore, chemical cross-linking of these macromers resulted in gels with increased stability compared to uncross-linked controls. These results demonstrate the feasibility of synthesizing poly( N-isopropylacrylamide)-based macromers that undergo tandem gelation and establish key criteria relating to the transition temperature and stability of these materials. The data suggest that these materials may be attractive substrates for tissue engineering and cellular delivery applications as the combination of mechanistically independent gelation techniques used in tandem may offer superior materials with regard to gelation kinetics and stability.

Injectable matrices and scaffolds for drug delivery in tissue engineering.

Injectable matrices and depots have been the subject of much research in the field of drug delivery. The classical tissue engineering paradigm includes a matrix or scaffold to facilitate tissue growth and provide structural support, cells, and the delivery of bioactive molecules. As both tissue engineering and drug delivery techniques benefit from the use of injectable materials due to the minimal invasiveness of an injection, significant crossover should be observed between injectable materials in both fields. This review aims to outline injectable materials and processing techniques used in both tissue engineering and drug delivery and to describe methods by which current injectable materials in the field of drug delivery can be adapted for use as injectable scaffolds for tissue engineering.

Review: mineralization of synthetic polymer scaffolds for bone tissue engineering.

It has repeatedly been shown that demineralization improves the ability of bone auto- and allografts to regenerate natural bone tissue. Conversely, much work in the field of bone tissue engineering has used composite materials consisting of a mineralized phase or materials designed to mineralize rapidly in situ. In this review, we seek to examine these disparate roles of mineralization and the underlying factors that cause this discordance and to examine methods and principles of the mineralization of synthetic polymer scaffolds. Biomimetic approaches to mineralization and phosphorus-containing materials are highlighted, and a brief section focusing on drug-delivery strategies using mineralized scaffolds is included.

Autologously generated tissue-engineered bone flaps for reconstruction of large mandibular defects in an ovine model.

The reconstruction of large craniofacial defects remains a significant clinical challenge. The complex geometry of facial bone and the lack of suitable donor tissue often hinders successful repair. One strategy to address both of these difficulties is the development of an in vivo bioreactor, where a tissue flap of suitable geometry can be orthotopically grown within the same patient requiring reconstruction. Our group has previously designed such an approach using tissue chambers filled with morcellized bone autograft as a scaffold to autologously generate tissue with a predefined geometry. However, this approach still required donor tissue for filling the tissue chamber. With the recent advances in biodegradable synthetic bone graft materials, it may be possible to minimize this donor tissue by replacing it with synthetic ceramic particles. In addition, these flaps have not previously been transferred to a mandibular defect. In this study, we demonstrate the feasibility of transferring an autologously generated tissue-engineered vascularized bone flap to a mandibular defect in an ovine model, using either morcellized autograft or synthetic bone graft as scaffold material.

Closure of large defects after microcystic lymphatic malformations using lateral intercostal artery perforator flap.

BACKGROUND AND AIM: The surgical treatment of microcystic lymphatic malformations (LMs) has historically been difficult and frustrating because of a high recurrence rate due to incomplete resection. However, complete removal of the multifocal and extensive lesions rely on accurate imaging diagnosis and effective repair methods for the resulting large defect. The purpose of this study was to repair large skin defects due to complete resection of microcystic LMs using lateral intercostal artery perforator (LICAP) flap. PATIENTS AND METHODS: Between January 2009 and June 2012, tissue defects in the axillary chest wall region of eight patients aged 13-22 years after microcystic LMs resections were closed using the LICAP flap. Flap donor sites in all patients were closed primarily except in one patient who underwent skin grafting. Before surgery, ultrasound and magnetic resonance imaging (MRI) examination were used to confirm the diagnosis and determine the scope and level of the abnormality for complete resection. RESULTS: All defects after microcystic LM excision were successfully closed using LICAP flaps. The follow-up period ranged from 1 to 3 years (mean 2.1 years). All flaps survived postoperatively. No recurrence occurred. Ultrasound and MRI follow-up also demonstrated flap survival without recurrence of microcystic LMs. No functional loss attributable to the LICAP flap harvest was identified in any case. CONCLUSIONS: Surgical resection is necessary for microcystic LMs. Imaging assists in the diagnosis and identification of the scope and level of lesions. The LICAP flap provides good coverage for the large defects and achieves acceptable morphology without functional deficits at flap donor sites. Ultrasound and MRI are safe and accurate diagnostic imaging methods for the pre- and postoperative evaluation of microcystic LMs in patients undergoing surgery.

Expansion of endothelial progenitor cells in high density dot culture of rat bone marrow cells.

In vitro expansion of endothelial progenitor cells (EPCs) remains a challenge in stem cell research and its application. We hypothesize that high density culture is able to expand EPCs from bone marrow by mimicking cell-cell interactions of the bone marrow niche. To test the hypothesis, rat bone marrow cells were either cultured in high density (2 × 10(5) cells/cm(2)) by seeding total 9 × 10(5) cells into six high density dots or cultured in regular density (1.6 × 10(4) cells/cm(2)) with the same total number of cells. Flow cytometric analyses of the cells cultured for 15 days showed that high density cells exhibited smaller cell size and higher levels of marker expression related to EPCs when compared to regular density cultured cells. Functionally, these cells exhibited strong angiogenic potentials with better tubal formation in vitro and potent rescue of mouse ischemic limbs in vivo with their integration into neo-capillary structure. Global gene chip and ELISA analyses revealed up-regulated gene expression of adhesion molecules and enhanced protein release of pro-angiogenic growth factors in high density cultured cells. In summary, high density cell culture promotes expansion of bone marrow contained EPCs that are able to enhance tissue angiogenesis via paracrine growth factors and direct differentiation into endothelial cells.

Salvage of infected left ventricular assist device with antibiotic beads.

BACKGROUND: The use of left ventricular assist devices has become common for the treatment of end-stage heart failure, both as a bridge to transplantation and as destination therapy. The nature of these devices and the comorbid conditions of the patients in whom the devices are implanted lead to high rates of device infection that are related directly to mortality. METHODS: Over 2 years, the senior author (S.A.I.) treated 26 patients with left ventricular assist device infections, ranging from superficial driveline infections to deeper pocket infections and device infections. An algorithm involving the use of repeated débridement and placement of antibiotic beads was used in treatment of these infections. Once cleared of infection, patients were treated with definitive closure or flap coverage of the formerly infected device component. RESULTS: Seventeen of 26 patients with left ventricular assist device-related infections were cleared of their infection using this method. Ten of these patients underwent flap coverage of the device after their infection was cleared. In patients that were cleared of infection, mortality was 29 percent, whereas patients with recalcitrant infections had a mortality of 67 percent over the course of the study. CONCLUSIONS: A systematic approach to treating left ventricular assist device-related infections has the potential to treat and clear these infections, with promising overall survival rates. This proposed algorithm led to high infection clearance rates compared with previously published literature. Infection clearance in patients on left ventricular assist device destination therapy may result in mortality rates approaching those of their uninfected peers.

Evaluation of antibiotic releasing porous polymethylmethacrylate space maintainers in an infected composite tissue defect model.

This study evaluated the in vitro and in vivo performance of antibiotic-releasing porous polymethylmethacrylate (PMMA)-based space maintainers comprising a gelatin hydrogel porogen and a poly(dl-lactic-co-glycolic acid) (PLGA) particulate carrier for antibiotic delivery. Colistin was released in vitro from either gelatin or PLGA microparticle loaded PMMA constructs, with gelatin-loaded constructs releasing colistin over approximately 7 days and PLGA microparticle-loaded constructs releasing colistin for up to 8 weeks. Three formulations with either burst release or extended release at different doses were tested in a rabbit mandibular defect inoculated with Acinetobacter baumannii (2×10(7) colony forming units ml(-1)). In addition, one material control that released antibiotic but was not inoculated with A. baumannii was tested. A. baumannii was not detectable in any animal after 12 weeks on culture of the defect, saliva, or blood. Defects with high dose extended release implants had greater soft tissue healing compared with defects with burst release implants, with 8 of 10 animals showing healed mucosae compared with 2 of 10 respectively. Extended release of locally delivered colistin via a PLGA microparticle carrier improved soft tissue healing compared with implants with burst release of colistin from a gelatin carrier.

Dispase rapidly and effectively purifies Schwann cells from newborn mice and adult rats.

In the present study, Schwann cells were isolated from the sciatic nerve of neonatal mice and purified using dispase and collagenase. Results showed that after the first round of purification with dispase, most of the Schwann cells appeared round in shape and floated in culture solution after 15 minutes. In addition, cell yield and cell purity were higher when compared to the collagenase group. After the second round of purification, the final cell yield for the dispase group was higher than that for the collagenase group, but no significant difference was found in cell purity. Moreover, similar results in cell quantity and purity were observed in adult Sprague-Dawley rats. These findings indicate that purification with dispase can result in the rapid isolation of Schwann cells with a high yield and purity.

Evaluation of bone regeneration using the rat critical size calvarial defect.

Animal models that are reliably reproducible, appropriate analogs to the clinical condition they are used to investigate, and that offer minimal morbidity and periprocedural mortality to the subject, are the keystone to the preclinical development of translational technologies. For bone tissue engineering, a number of small animal models exist. Here we describe the protocol for one such model, the rat calvarial defect. This versatile model allows for evaluation of biomaterials and bone tissue engineering approaches within a reproducible, non-load-bearing orthotopic site. Crucial steps for ensuring appropriate experimental control and troubleshooting tips learned through extensive experience with this model are provided. The surgical procedure itself takes ∼30 min to complete, with ∼2 h of perioperative care, and tissue collection is generally performed 4-12 weeks postoperatively. Several analytical techniques are presented, which evaluate the cellular and extracellular matrix components, functionality and mineralization, including histological, mechanical and radiographic methods.

In situ formation of porous space maintainers in a composite tissue defect.

Reconstruction of composite defects involving bone and soft tissue presents a significant clinical challenge. In the craniofacial complex, reconstruction of the soft and hard tissues is critical for both functional and aesthetic outcomes. Constructs for space maintenance provide a template for soft tissue regeneration, priming the wound bed for a definitive repair of the bone tissue with greater success. However, materials used clinically for space maintenance are subject to poor soft tissue integration, which can result in wound dehiscence. Porous materials in space maintenance applications have been previously shown to support soft tissue integration and to allow for drug release from the implant to further prepare the wound bed for definitive repair. This study evaluated solid and low porosity (16.9% ± 4.1%) polymethylmethacrylate space maintainers fabricated intraoperatively and implanted in a composite rabbit mandibular defect model for 12 weeks. The data analyses showed no difference in the solid and porous groups both histologically, evaluating the inflammatory response at the interface and within the pores of the implants, and grossly, observing the healing of the soft tissue defect over the implant. These results demonstrate the potential of porous polymethylmethacrylate implants formed in situ for space maintenance in the craniofacial complex, which may have implications in the potential delivery of therapeutic drugs to prime the wound site for a definitive bone repair.

Gradual pore formation in natural origin scaffolds throughout subcutaneous implantation.

This study used a rat subcutaneous implantation model to investigate gradual in situ pore formation in a self-regulating degradable chitosan-based material, which comprises lysozyme incorporated into biomimetic calcium phosphate (CaP) coatings at the surface to control the scaffold degradation and subsequent pore formation. Specifically, the in vivo degradation of the scaffolds, the in situ pore formation, and the tissue response were investigated. Chitosan or chitosan/starch scaffolds were studied with and without a CaP coating in the presence or absence of lysozyme for a total of six experimental groups. Twenty-four scaffolds per group were implanted, and eight scaffolds were retrieved at each of three time points (3, 6, and 12 weeks). Harvested samples were analyzed for weight loss, microcomputed tomography, and histological analysis. All scaffolds showed pronounced weight loss and pore formation as a function of time. The highest weight loss was 29.8% ± 1.5%, obtained at week 12 for CaP chitosan/starch scaffolds with lysozyme incorporated. Moreover, all experimental groups showed a significant increase in porosity after 12 weeks. At all time points no adverse tissue reaction was observed, and as degradation increased, histological analysis showed cellular ingrowth throughout the implants. Using this innovative methodology, the ability to gradually generate pores in situ was clearly demonstrated in vivo.