Visible light photoinitiation of mesenchymal stem cell-laden bioresponsive hydrogels.

Biological activity can be added to synthetic scaffolds by incorporating functional peptide sequences that provide enzyme-mediated degradation sites, facilitate cellular adhesion or stimulate signaling pathways. Poly(ethylene glycol) diacrylate is a popular synthetic base for tissue engineering scaffolds because it creates a hydrophilic environment that can be chemically manipulated to add this biological functionality. Furthermore, the acrylate groups allow for encapsulation of cells using photopolymerization under physiological conditions. One complication with the addition of these peptides is that aromatic amino acids absorb light at 285 nm and compete with the ultraviolet (UV)-sensitive photoinitiators such as IrgacureTM 2959 (I2959), the most commonly used initiator for cytocompatible photoencapsulation of cells into synthetic scaffolds. In this study we define non-toxic conditions for photoencapsulation of human mesenchymal stem cells (hMSC) in PEGDA scaffolds using a visible light photoinitiator system composed of eosin Y, triethanolamine and 1-vinyl-2-pyrrolidinone. This visible light photoinitiator produced hydrogel scaffolds with an increased viability of encapsulated hMSCs and a more tightly crosslinked network in one-third the time of UV polymerization with I2959.

3D Culture Facilitates VEGF-Stimulated Endothelial Differentiation of Adipose-Derived Stem Cells.

De novo vascularization of implantable tissue and whole organ constructs has been a significant challenge in the field of tissue engineering; the use of endothelial cell populations for this task is constrained by the cell population's limited regeneration capacity and potential for loss of function. Thus, there is a need for a stem-cell population that may be induced into an endothelial cell phenotype reliably. Adipose derived stem cells (ADSCs) are multipotent cells that can be readily isolated from donor fat and may have the potential to be readily induced into endothelial cells. The ability to stimulate endothelial differentiation of these cells has been limited in standard 2D culture. We hypothesized that 3D culture would yield better differentiation. To study the influence of cell density and culture conditions on the potential of ADSCs to differentiate into an endothelial-like state, we seeded these cells types within a 3D cell-adhesive, proteolytically degradable, peptide-modified poly(ethylene-glycol) (PEG) hydrogel. ADSCs were either cultured in basal media or pro-angiogenic media supplemented with 20 ng/mL of VEGF in 2D and then encapsulated at low or high densities within the PEG-based hydrogel. These encapsulated cells were maintained in either basal media or pro-angiogenic media. Cells were then isolated from the hydrogels and cultured in Matrigel to assess the potential for tubule formation. Our work shows that maintenance of ADSCs in a pro-angiogenic medium in 2D monoculture alone does not result in any CD31 expression. Furthermore, the level of CD31 expression was affected by the density of the cells encapsulated within the PEG-based hydrogel. Upon isolation of these cells, we found that these induced ADSCs were able to form tubules within Matrigel, indicative of endothelial function, while ADSCs cultured in basal medium could not. This finding points to the potential for this stem-cell population to serve as a safe and reliable source of endothelial cells for tissue engineering and regenerative medicine purposes.

Blood vessel matrix: a new alternative for abdominal wall reconstruction.

BACKGROUND: Biologic matrices offer a new approach to the management of abdominal wall defects when the use of other foreign material is not ideal. A member of our team (GEA) developed a biological decellularized matrix generated from harvested blood vessels of swine blood vessel matrix (BVMx). The aim of our study was to investigate whether this novel collagen-based biological matrix is safe and effective for the repair of abdominal wall hernia defects in a rat model. METHODS: Full thickness abdominal wall defects were created in rats and repaired with our BVMx. After implantation as an underlay for 30 and 90 days, animals were sacrificed and the implanted material evaluated for herniation, adhesions, breaking strength, inflammation, and revascularization. RESULTS: No evidence of herniation was noted at 30 (n = 7) or 90 (n = 7) days after repair. Adhesions, if present, were filmy and easily separated. The mean area of visceral adhesions to the BVMx was 18.9 +/- 11.0% at 30 days and 7.1 +/- 3.1% at 90 days post implantation (P = 0.33). The breaking strength of the BVMx-fascial interface was 4.5 +/- 0.8 N at 30 days and 4.5 +/- 2.4 N at 90 days post implantation (P = 0.98). Histologic analysis demonstrated that the BVMx elicited a mild transient inflammatory response and supported fibroblast migration, deposition of newly formed collagen, and neovascularization. CONCLUSIONS: These data confirm that this BVMx supports vascular ingrowth and provides adequate strength for the repair of abdominal wall defects. Future studies in a large animal model are required to assess its validity for human application.

Applications of nanotechnology to biotechnology commentary.

The ability to systematically modify the properties of nanostructures by controlling their structure and their surface properties at a nanoscale level makes them extremely attractive candidates for use in biological contexts, from fundamental scientific studies to commercially viable technologies.

Nitric oxide-generating polymers reduce platelet adhesion and smooth muscle cell proliferation.

We have developed polymeric biomaterials capable of providing localized and sustained production of nitric oxide (NO) for the prevention of thrombosis and restenosis. In the current study, we have characterized the kinetics of NO production by these materials and investigated their efficacy in reducing platelet adhesion and smooth muscle cell proliferation in vitro. Three nitric oxide donors with different half-lives were covalently incorporated into photopolymerized polyethylene glycol hydrogels. Under physiological conditions, NO was produced by these hydrogels over periods ranging from hours to months, depending upon the polymer formulation. NO production was inhibited at acidic pH, which may be useful for storage of the materials. The NO-releasing materials successfully inhibited smooth muscle cell growth in culture. Platelet adhesion to collagen-coated surfaces was also inhibited following exposure of whole blood to NO-producing hydrogels. The effects of NO production by these hydrogels on platelet adhesion and the proliferation of smooth muscle cells suggest that these materials could reduce thrombosis and restenosis following procedures such as balloon angioplasty.

Comparison of covalently and physically cross-linked polyethylene glycol-based hydrogels for the prevention of postoperative adhesions in a rat model.

A covalently and a physicochemically cross-linked hydrogel, both based primarily on polyethylene glycol and both formed in situ, were compared side by side in a rat uterine horn devascularization and serosal injury model for efficacy in adhesion prevention. The primary difference between the two materials was the nature of their cross-linking. The covalently cross-linked hydrogel was a photopolymerized polyethylene glycol-co-lactic acid diacrylate, and the physically cross-linked hydrogel was a polyethylene glycol-co-polypropylene glycol, Poloxamer 407. In the surgical model employed, application of the covalently cross-linked hydrogel reduced the extent of adhesion formation from 75 +/- 10% in the control group to 16 +/- 6% (mean +/- s.d., P < 0.001). Application of the physically cross-linked hydrogel reduced adhesion formation to 38 +/- 19% (P < 0.01). Retention of the two hydrogels upon the site of application was also evaluated. The covalently cross-linked hydrogel formed a continuous barrier upon the uterine horns for more than 4 d, while the physicochemically cross-linked hydrogel was present upon the uterine horns for less than 2 d. This difference in retention was probably the cause of the difference in efficacy and may be attributed to the nature of the cross-linking.

Tethered-TGF-beta increases extracellular matrix production of vascular smooth muscle cells.

Biomaterials developed for tissue engineering and wound healing applications need to support robust cell adhesion, yet also need to be replaced by new tissue synthesized by those cells. In order to maintain mechanical integrity of the tissue, the cells must generate sufficient extracellular matrix before the scaffold is degraded. We have previously shown that materials containing cell adhesive ligands to promote or improve cell adhesion can decrease extracellular matrix production (Mann et al., Modification of surfaces with cell adhesion peptides alters extracellular matrix deposition. Biomaterials 1999;20:2281-6). Such decreased matrix production by cells in tissue engineering scaffolds may result in tissue failure. However, we have found that TGF-beta1 can be used in scaffolds to dramatically increase matrix production. Matrix production by vascular smooth muscle cells grown on adhesive ligand-modified glass surfaces and in PEG hydrogels containing covalently bound adhesive ligands was increased in the presence of 0.04 pmol/ml (1 ng/ml) TGF-beta1. TGF-beta1 can counteract the effect of these adhesive ligands on matrix production; matrix production could be increased even above that observed in the absence of adhesive peptides. Further, TGF-beta1 covalently immobilized to PEG retained its ability to increase matrix production. Tethering TGF-beta1 to the polymer scaffold resulted in a significant increase in matrix production over the same amount of soluble TGF-beta1.

Platelet adhesion on a bioresorbable poly(propylene fumarate-co-ethylene glycol) copolymer.

Platelet adhesion and aggregation on poly(propylene fumarate-co-ethylene glycol), P(PF-co-EG), hydrogels was examined under both static and flow conditions. Adherent platelets were quantified under static conditions using both 111Indium oxine-labeled platelets as well as a lactate dehydrogenase, LDH, assay. The radiolabeling assay showed a significant decrease in platelet attachment on the copolymer hydrogel films relative to the poly(propylene fumarate), PPF, homopolymer. In addition, there were reductions in adhesion resulting from the increase in poly(ethylene glycol), PEG, weight percent or molecular weight. There was good agreement between both assays under static conditions for the copolymer films. Platelet surface coverage was quantified under flow conditions in a parallel plate flow chamber using the LDH assay. There was a dramatic decrease in the number of adherent platelets on the copolymers relative to glass and silicone rubber controls. All of the copolymer surfaces showed minimal aggregation with no thrombus formation or platelet spreading as assessed qualitatively using scanning electron microscopy. These results suggest that P(PF-co-EG) is a good candidate for development as a cardiovascular implant.

Modification of surfaces with cell adhesion peptides alters extracellular matrix deposition.

The goal of the current study was to evaluate matrix protein synthesis by cells cultured on materials that had been modified with cell adhesion ligands. We examined the effects of surface peptide density and of peptides with different affinities on the extracellular matrix production of smooth muscle cells, endothelial cells and fibroblasts. While initial adhesion was greatest on the higher density peptide surfaces, all cell types exhibited decreased matrix production on the more highly adhesive surfaces. Similarly, when different peptides were evaluated, matrix production was the lowest on the most adhesive surface and highest on the least adhesive surface. These results suggest that extracellular matrix synthesis may be regulated, to some extent, by signal transduction initiated by adhesion events. This may pose limitations for use of bioactive materials as tissue engineering scaffolds, as matrix production is an important aspect of tissue formation. However, it may be possible to increase matrix production on highly adhesive surfaces using exogenous factors. TGF-beta was shown to increase matrix production by both smooth muscle cells and endothelial cells.

Smooth muscle cell growth in photopolymerized hydrogels with cell adhesive and proteolytically degradable domains: synthetic ECM analogs for tissue engineering.

Photopolymerizable polyethylene glycol (PEG) derivatives have been investigated as hydrogel tissue engineering scaffolds. These materials have been modified with bioactive peptides in order to create materials that mimic some of the properties of the natural extracellular matrix (ECM). The PEG derivatives with proteolytically degradable peptides in their backbone have been used to form hydrogels that are degraded by enzymes involved in cell migration, such as collagenase and elastase. Cell adhesive peptides, such as the peptide RGD, have been grafted into photopolymerized hydrogels to achieve biospecific cell adhesion. Cells seeded homogeneously in the hydrogels during photopolymerization remain viable, proliferate, and produce ECM proteins. Cells can also migrate through hydrogels that contain both proteolytically degradable and cell adhesive peptides. The biological activities of these materials can be tailored to meet the requirements of a given tissue engineering application by creating a mixture of various bioactive PEG derivatives prior to photopolymerization.

Prevention of postoperative adhesions in the rat by in situ photopolymerization of bioresorbable hydrogel barriers.

OBJECTIVE: To assess the efficacy of a novel resorbable hydrogel barrier for preventing postoperative adhesions in animals. METHODS: A hydrogel barrier was formed in situ by photopolymerizing a solution of a macromolecular prepolymer in buffered saline using long-wavelength ultraviolet light. Two models in the rat were evaluated. In a primary adhesion model, devascularization and serosal injury were performed on the uterine horns using bipolar electrocautery. The prepolymer solution was applied to the horns and illuminated to form the barrier. On the seventh postoperative day, the fraction of the length of the horns involved in adhesions was scored, as was the quality of the adhesions. In a readhesion model, adhesions were formed as described and were surgically lysed on the seventh day, then were treated subsequently with the barrier and scored after 7 additional days. Each group in both models consisted of seven animals per treatment condition. Four prepolymer concentrations were examined in the primary adhesion model, and the optimal one was examined in the readhesion model. RESULTS: A conformal hydrogel barrier coating was formed upon in situ photopolymerization and adhered to the treated tissues. No residual hydrogel barrier was observed 7 days after application of the optimal gel concentrations. In the primary adhesion model, the mean fraction of the horns involved in adhesions was reduced significantly, from 76% in controls to 10% (P < .0001), and treatment with a 10% solution of prepolymer was determined to be optimal (P = .025). In the readhesion model, surgical lysis of adhesions alone did not reduce adhesions significantly (from 86% to 79%; P = .3), whereas lysis with barrier treatment did (79% to 28%; P = .002). CONCLUSIONS: In situ photopolymerization allowed the formation of adherent, conformal barriers, which demonstrated high efficacy in the prevention of adhesion formation and reformation in animals. This efficacy and ease of use warrant clinical evaluation.

Efficacy of a resorbable hydrogel barrier, oxidized regenerated cellulose, and hyaluronic acid in the prevention of ovarian adhesions in a rabbit model.

OBJECTIVE: To compare the efficacy of a resorbable hydrogel material with oxidized regenerated cellulose and hyaluronic acid in an ovarian adhesion model. DESIGN: Controlled, blinded, and randomized study involving female rabbits. SETTING: Academic research environment. INTERVENTIONS: A water-soluble hydrogen precursor was applied to the ovary as a liquid and converted to a hydrogel by exposure to long wavelength ultraviolet light, a 0.4% solution of hyaluronic acid was applied to the ovary, or an oxidized regenerated cellulose patch was applied to the ovary after wedge resectioning. MAIN OUTCOME MEASURES: Extent and severity of adhesion formation. RESULTS: Application of the hydrogel reduced adhesion formation by 88%. Neither oxidized regenerated cellulose nor hyaluronic acid reduced adhesion formation. CONCLUSION: The photopolymerized, resorbable hydrogel material is highly effective for the reduction of periovarian adhesions in this model.

Results of spinal arthrodesis with pedicle screw-plate fixation.

Sixty-one patients were followed for an average of thirty months (range, twenty-four to thirty-five months) after arthrodesis of the lumbar or lumbosacral spine with pedicle screw-plate fixation for painful degenerative arthritis, spondylolisthesis, or pseudarthrosis. The patients rated the clinical result according to an analog scale. Most patients reported a marked decrease in pain and an increase in function, and two-thirds were able to work full time. The result of the operation was regarded as a clinical failure if the patient considered it so, if an additional operation had been done, or if the functional and pain scores were not good (that is, if the patient was not able to work full time and the rating for pain was more than 5 of 10 points). Seventeen (28 per cent) of the patients were considered to have a clinical failure, with the lowest rate (20 per cent) for patients who had painful degenerative arthritis and the highest rate (47 per cent) for patients who had had a pseudarthrosis before the operation. The rate of fusion was 90 per cent in patients who had painful degenerative disease, 93 per cent in patients who had spondylolisthesis, and 65 per cent in patients who had had a pseudarthrosis preoperatively.

Comparison of insufflation vs. retractional technique for laparoscopic-assisted intervertebral fusion of the lumbar spine.

Laparoscopic transperitoneal fusion of the L5-S1 spinal interspace has become a common procedure. Retroperitoneal retraction and laparoscopic instrumentation without insufflation also allows visualization of the upper lumbar spaces, but this procedure is much more difficult to accomplish. We review and compare our results using each of these techniques for the treatment of mechanical instability and chronic back pain. A total of 35 selected patients underwent intervertebral fusion between February 1996 and August 1998. Their mean age was 48 years. There were 22 female and 13 male patients. Standard CO2 insufflation was used in 10 patients with L5-S1 fusions. Retractional gasless technique was used in nine patients with fusions at L5-S1, 16 patients at L4-L5, one patient at L3-L4, three patients at L2-3, and one patient at L1-L2. Thus, we performed a total of 40 lumbar fusions in 35 patients. In the 19 patients with the gasless technique, a balloon dissector and retractor facilitated the retroperitoneal exposure. Seven of these 19 patients were converted to open procedures, most commonly due to lacerations of the peritoneal lining that prohibited visualization. None of the L5-S1 patients with insufflation were converted to open. Mean operative time in the insufflated patients was 152 min vs. 181 min for the retractional technique. There were seven complications in the transperitoneal group: one fusion device migration, one postoperative UTI, one intracerebral hemorrhage, one severe postoperative pancreatitis, and three iliac vein lacerations. There were 16 complications in the retroperitoneal group: one deep vein thromboses, one serosal bowel injury, one small tear in the spleen, one cage migration, one postoperative pulmonary atelectasis, one postoperative hydrocele, four postoperative ileus, and six peritoneal tears. The mean postoperative stay was three days for both groups. There were no deaths. The L5-S1 interspace is best approached transperitoneally for anterior fusion. Although the retroperitoneal retractional technique is much more difficult and has a longer and steeper learning curve, it does allow laparoscopic anterior fusion of the upper lumbar spine.

Incidence of deep vein thrombosis in major adult spinal surgery.

Deep vein thrombosis and pulmonary embolus are well known complications of lengthy orthopaedic procedures. Historically spine surgery has been associated with few thrombotic complications. Recent widespread use of instrumentation in adult cases led to concern regarding the incidence of deep vein thrombosis and pulmonary embolus. Forty-one patients underwent posterior spinal procedures using either pedicular or segmental instrumentation. One day before discharge all patients underwent noninvasive testing using color duplex Doppler imaging to rule out deep vein thrombosis. Six patients were noted to have tests results compatible with deep vein thrombosis. The incidence of deep vein thrombosis during this study (14%) reveals a group of patients who would have been discharged with ongoing thrombosis and potential pulmonary embolism.

Complications of the variable screw plate pedicle screw fixation.

In this study, 124 consecutive cases of posterior spinal fusion with variable screw plate fixation were reviewed. In 33 patients (27%), 41 complications were identified. Urinary tract infection without sequelae developed in 13 patients. Dural tear occurred in seven patients and wound hematoma in five. Wound infection developed in three patients; one was subfascial requiring instrument and graft removal. Neurologic deficit developed in seven patients (6%), in five of whom the deficit was due to manipulation and reduction of neural elements. Two of the seven deficits were believed to be caused by misplaced pedicle screws. Variable screw plate fixation is a formidable procedure with a significant complications rate.

Management of retroperitoneal chylous leakage.

Lymphatic complications are uncommon after spinal surgery. Postsurgical collections of lymph may result in chyloma, chylothorax, chylous ascites, or chyloretroperitoneum. Recommended treatment of chylothorax or chylous ascites includes drainage, and a low-fat diet using medium-chain triglyceride or total parenteral nutrition. The only reported case of chyloretroperitoneum concluded with a recommendation for retroperitoneal drainage. The authors present a series of three patients who underwent anterior spinal procedures complicated by retroperitoneal lymphatic injury. All three were treated without retroperitoneal drains, and at follow-up, none has shown any adverse sequelae from this method of treatment. The authors disagree with past recommendations for drainage of the retroperitoneal space because this may prolong drainage and deplete nutrition.

The morbidity of heparin therapy after development of pulmonary embolus in patients undergoing thoracolumbar or lumbar spinal fusion.

STUDY DESIGN: The postoperative course of patients who developed a pulmonary embolus after thoracolumbar or lumbar spinal fusion treated with heparin was studied to quantify the morbidity risk of anticoagulation. OBJECTIVE: To compare the morbidity risk of heparinization with that of an alternative form of therapy--inferior vena cava filter placement. SUMMARY OF BACKGROUND DATA: Therapeutic heparinization was the current treatment of choice for patients who develop thromboembolic disease after surgery. Although heparin usage was reported to be associated with a number of complications after other orthopedic and general surgical procedures, no information was available to identify complications of heparinization after lumbar or thoracolumbar spine surgery or to define the risk of such complications. METHODS: Twenty-two members of the Scoliosis Research Society were polled to determine their experiences with the anticoagulation of this subset of patients. Surgeons polled had a combined experience of 250 man-years and had performed more than 13000 thoracolumbar and lumbar spinal fusions. The MEDLINE database was used to review pertinent English language publications describing inferior vena cava filter complications, effectiveness, safety, and indications for use. RESULTS: Nine patients were located who fit the inclusion criteria of this study. Six (67%) had complications attributable to heparinization. Clinically significant complications of filter placement ranged from 0.12% to 10.1%. CONCLUSIONS: Heparinization after the development of pulmonary embolus in patients recently undergoing spinal fusion is associated with a high complication rate. The morbidity of vena cava filter placement is low and should be considered a treatment alternative in the treatment of patients who experience pulmonary embolus after surgery.

Hidden photoalignment of liquid crystals in the isotropic phase.

We found the effect of a hidden photoalignment of a dye-doped nematic liquid crystal (LC) on a nonphotosensitive polymer surface after polarized irradiation of the cell in the isotropic phase. We observed that irradiation resulted in a uniform planar orientation of the LC after cooling to the mesophase. The direction of a light-induced easy axis on the polymer can be either parallel or perpendicular to the polarization of the incident light, depending on the light intensity. We attribute this behavior to two mechanisms of photoalignment: light-induced adsorption of dye molecules on the substrate, and anisotropic desorption in a previously adsorbed dye layer. The experimental results on photoalignment of a LC on a thin dye film confirm our model.

Photo-orientation of liquid crystals due to light-induced desorption and adsorption of dye molecules on an aligning surface.

We show that adsorption of dye molecules control the light-induced alignment of dye-doped nematic liquid crystal (LC) on a nonphotosensitive polymer surface. The dependencies of light-induced twist structures on exposure, thermal baking, thickness, and aging before irradiation of the LC cells allowed us to propose the following mechanism for the alignment. Before irradiation, the "dark"-adsorbed layer on the tested surface is formed from dye molecules predominantly aligned along the initial direction of the director. Irradiation of the cell with linearly polarized light produces an additional layer with different orientational ordering of dye molecules. The final easy axis is determined by the competition of "dark" and light-induced contributions to anchoring and is aligned between the "dark" easy axes and polarization of the light. For quantitative interpretation, we apply the tensor model of anchoring and assume that the photoalignment in the mesophase is a cumulative effect of the light-induced anchoring on the background of the already existing anisotropic "dark" dye layer.