Silk fibroin, gelatin, and human placenta extracellular matrix-based composite hydrogels for 3D bioprinting and soft tissue engineering.

BACKGROUND: There is a great clinical need and it remains a challenge to develop artificial soft tissue constructs that can mimic the biomechanical properties and bioactivity of natural tissue. This is partly due to the lack of suitable biomaterials. Hydrogels made from human placenta offer high bioactivity and represent a potential solution to create animal-free 3D bioprinting systems that are both sustainable and acceptable, as placenta is widely considered medical waste. A combination with silk and gelatin polymers can bridge the biomechanical limitations of human placenta chorion extracellular matrix hydrogels (hpcECM) while maintaining their excellent bioactivity. METHOD: In this study, silk fibroin (SF) and tyramine-substituted gelatin (G-TA) were enzymatically crosslinked with human placental extracellular matrix (hpcECM) to produce silk-gelatin-ECM composite hydrogels (SGE) with tunable mechanical properties, preserved elasticity, and bioactive functions. The SGE composite hydrogels were characterized in terms of gelation kinetics, protein folding, and bioactivity. The cyto- and biocompatibility of the SGE composite was determined by in vitro cell culture and subcutaneous implantation in a rat model, respectively. The most cell-supportive SGE formulation was then used for 3-dimensional (3D) bioprinting that induced chemical crosslinking during extrusion. CONCLUSION: Addition of G-TA improved the mechanical properties of the SGE composite hydrogels and inhibited crystallization and subsequent stiffening of SF for up to one month. SGE hydrogels exhibit improved and tunable biomechanical properties and high bioactivity for encapsulated cells. In addition, its use as a bioink for 3D bioprinting with free reversible embedding of suspended hydrogels (FRESH) has been validated, opening the possibility to fabricate highly complex scaffolds for artificial soft tissue constructs with natural biomechanics in future.

Extract derived from rat brains in the acute phase following traumatic brain injury impairs survival of undifferentiated stem cells and induces rapid differentiation of surviving cells.

Dramatic cerebral responses following brain injury (TBI) comprise inflammation, cell death, and modulation of trophic factor release. These cerebral modulations might induce and/or attenuate acute neuronal damage. Here, we investigated the effect of tissue extract derived from healthy (HBE) or injured rat brain (TBE) on the differentiation of cultured embryonic stem cells in vitro. Rats were sacrificed at t = 45 minutes following lateral fluid-percussion injury and extracts of cerebral tissue were prepared from 4-6 healthy or injured rat brain hemispheres. Murine embryonic stem cells (CGR8) cultured in serum-free medium were then conditioned for a week with HBE or TBE. Omission of serum from the culture medium induced neural differentiation of CGR8 stem cells, as indicated by a significant time dependent down-regulation of oct-4 with a concomitant upregulation of nestin after 7 days. In parallel cell loss was observed that seemed to be largely due to apoptotic cell death. In TBE treated cells, on the other hand, a significant amplification of apoptotic cell death, enhancement of nestin and MAP2 expression and marked morphological changes such as axonal-like outgrowth was observed within 3 days of conditioning. Treatment of stem cells with HBE resulted in less pronounced neuronal differentiation processes. Axonal-like outgrowth was not observed. Our data suggest that during the early acute phase of traumatic injury the cerebral environment is disposed to detrimental as well as potent protective signals that seem to rapidly induce neurogenic processes.

Developmental potential of the murine embryonic stem cells transplanted into the healthy rat brain--novel insights into tumorigenesis.

Although engraftment of undifferentiated pluripotent embryonic stem cells (ESCs) into the injured central nervous system (CNS) may lead to targeted cell replacement of lost/damaged cells, sustained proliferative activity combined with uncontrolled differentiation of implanted cells presents a risk of tumor formation. As tumorigenic potential is thought to be associated with pluripotency of embryonic stem cells, pre-differentiation may circumvent this problem. Recently, it has been demonstrated that tumorigenesis occurs despite pre-differentiation if the neural precursor cells are implanted into the brain of a homologous animal (e.g., mouse to mouse). However, xenotransplantation (e.g., mouse to rat) without pre-differentiation, lead to the development of healthy neuronal cells, in absence of tumor formation, suggesting that tumor-suppressive effects of host tissue on engrafted ESCs may play a role in transplant tumorigenesis. We critically investigated tumorigenesis and possible mechanisms of anticipated tumor-suppressive effect under conditions analogous to previously published studies. Xenotransplantation of D-3 murine ESCs into uninjured adult rat brains lacking any preliminary inflammatory potential was found to lead to tumor formation in 5 out of 8 of animals within 2 weeks postimplantation. Tumor-suppressive effects, reflected by Erdo et. al could possibly be ascribed to immunomodulatory activity of macrophages scavenging the tumorigenic fraction of the implanted cells. The importance of number of engrafted cells, implantation site and immunosuppressive effects are discussed as possible variables determining tumorigenic outcome after ESC transplantation.

Embryonic stem cell transplantation after experimental traumatic brain injury dramatically improves neurological outcome, but may cause tumors.

Transplantation of embryonic stem (ES) cells may provide cures for the damaged nervous system. Pre-differentiated ES or neuronal precursor cells have been investigated in various animal models of neurodegenerative diseases including traumatic brain injury (TBI). To our knowledge, no study has yet examined the effects of undifferentiated, murine ES cells on functional recovery and tumorigenity following implantation into injured rat brains. We evaluated the effect of transplantation of undifferentiated, murine embryonic cells on the recovery of motor function following lateral fluid percussion brain injury in Sprague-Dawley rats. At 3 days post-injury, animals received stereotactic injections of either embryonic stem cell suspension or injections of phosphate buffered saline without cells (control) into the injured cortex. Neurological motor function assessments were performed before injury, 72 h, 1, 3, and 6 weeks after transplantation using a Rotatrod and a Composite Neuroscore test. During this time period brain injured animals receiving ES cell transplantation showed a significant improvement in the Rotarod Test and in the Composite Neuroscore Test as compared to phosphate buffered saline (PBS)-treated animals. At 1 week post-transplantation, ES cells were detectable in 100% of transplanted animals. At 7 weeks following transplantation, EScells were detectable in only one animal. Two of 10 xenotransplanted animals revealed tumor formation over the observation period. These findings provide evidence for therapeutic potency of embryonic stem cell transplantation after TBI in rat, but also raise serious safety concerns about the use of such cells in human.

Trauma-associated inflammatory response impairs embryonic stem cell survival and integration after implantation into injured rat brain.

Pluripotent embryonic stem cells were shown to survive and differentiate into mature neuronal cells after implantation in experimental models of Parkinson disease and cerebral ischemia. Embryonic stem cell transplantation has also been proposed as a potential therapy for cerebral trauma, characteristic of massive loss of multiple cell types due to primary insult and secondary sequelae. Green fluorescent protein (GFP)-transfected murine embryonic stem cells were implanted into the ipsi or contralateral cortex of male Sprague-Dawley rats 72 h after fluid-percussion injury. Animals were sacrificed at day 5 or week 7 postimplantation. Brain sections were examined using conventional and fluorescent double-labelling immunohistochemistry. Five days after implantation, clusters of GFP-positive cells undergoing partial differentiation along neuronal pathway, were detected at the implantation site. However, after 7 weeks, only a few GFP-positive cells were found, indicating an extensive loss of stem cells during this time period. For the first time, we proved the observed cell loss to be mediated via phagocytosis of implanted cells by activated macrophages. Cerebral trauma, induced 3 days prior to implantation, has activated the inflammatory potential of otherwise immunologically privileged tissue. Subsequent cell implantation was accompanied by reactive astrogliosis, activation of microglia, as well as a massive invasion of macrophages into transplantation sites even if the grafts were placed into contralateral healthy hemispheres, remote from the traumatic lesion. Our results demonstrate a significant post-traumatic inflammatory response, which impairs survival and integration of implanted stem cells and has generally not been taken into account in designs of previous transplantation studies.

Functional treatment and early weightbearing after an ankle fracture: a prospective study.

OBJECTIVE: Postoperative care for ankle fractures is generally 1 of 2 regimens: 1) functional treatment combined with early weightbearing (EWB), or 2) immobilization in a cast/orthosis for 6 weeks without weightbearing (6WC). The objective of this study was 2-fold: 1) to follow a prospective group treated with EWB as to long-term subjective and objective outcomes, and 2) to compare a subset of this group with a matched group of historic controls treated with 6WC. DESIGN: Prospective, clinical, cohort observation, and retrospective matched pair analysis. SETTING: University hospital, level 1 trauma center. PATIENTS: Forty-three patients (20 males; mean age, 49 +/- 14 years) with operated Weber B/C fractures underwent EWB. For comparison, 23 patients of this group were matched to a same number of historic controls with respect to age, gender, body mass index, and fracture type. INTERVENTION: Open reduction and internal fixation (ORIF) using a 1/3-tubular-fibula-plate for the fibula, and malleolar screws for the medial malleolus fracture (in cases with a bimalleolar ankle fracture) followed by EWB or 6WC. MAIN OUTCOME MEASUREMENTS: Olerud and Tegner scores at follow-up (at least 12 months after surgery), time to full weightbearing, return to work, pain intensity (numerical rating scale (NRS)), and hospital stay. Statistical comparisons were performed by using the Mann-Whitney U test or Fisher exact test (P < 0.05). RESULTS: Patients with EWB were full weightbearing at 7 +/- 3 weeks and returned to work at 8 +/- 5 weeks after surgery. At follow-up (mean, 20 +/- 11 months after surgery), all EWB patients showed good results in the Olerud score (90 +/- 13 points). Matched-pair analysis in 23 patients in each group revealed differences between EWB and 6WC groups for hospital stay (mean, 10.8 +/- 4.7 vs. 13.6 +/- 6 days; P = 0.12), time to full weightbearing (mean, 7.7 +/- 3.1 vs. 13.5 +/- 9.4 weeks; P = 0.01), and time until return to work (mean 9.2 +/- 5.5 vs. 10.8 +/- 7 weeks; P = 0.63). No differences concerning pain intensities were observed (EWB vs. 6WC: NRS = 1.9 vs. 1.7; P = 0.12). At follow-up, Olerud scores were generally considered good for both groups; however, mean values in EWB patients were slightly higher (87 +/- 14 vs. 79 +/- 19 points; P = 0.25). In both groups, the majority of patients reached their preinjury level of activity as demonstrated by Tegner scores. CONCLUSIONS: EWB patients tolerated earlier full weightbearing compared with 6WC patients, and there were no disadvantages with EWB compared with 6WC concerning hospital stay, pain intensities, time until return to work, and Olerud/Tegner Scores. Potential candidates for EWB are patients with a stable osteosynthesis of their fractured ankles as judged by the responsible surgeon, compliance, and high motivation.

Characterization of a new rat model of experimental combined neurotrauma.

Traumatic brain injury (TBI) is present in two-thirds of patients with multiple injuries and in one-third combined with injuries of the extremities. Studies on interactive effects between central and peripheral injuries are scarce due to the absence of clinically relevant models. To meet the demand for "more-hit" models, an experimental model of combined neurotrauma (CNT) incorporating a standardized TBI via lateral fluid percussion (LFP) together with a peripheral bone fracture, i.e., tibia fracture, is introduced. Sprague-Dawley rats were randomized to four experimental groups: controls (n = 10), animals with TBI (n = 30), animals with tibia fracture (n = 30), and animals with CNT (n = 30). Morphological aspects of brain and bone injury were analyzed via standard histopathological procedures and x-ray. Trauma-induced neuromotor dysfunction was assessed using a standardized neuroscore. For interactive effects between injuries, we studied the extent and temporal pattern of circulating interleukin 6 (IL-6) levels via immunoassay and callus formation at fracture sites by means of microradiography. LFP produced an ipsilateral lesion with cortical contusion, hemorrhage, mass shift, and neuronal cell loss (adjacent cortex and hippocampus CA-2/-3), along with contralateral neuromotor dysfunction. X-rays confirmed complete fractures in the middle of the bone shaft. The type of injury (P < 0.001) and time (P = 0.022) were significantly associated with increased IL-6 levels. CNT produced the highest IL-6 plasma levels with a maximum peak at 6 h after trauma (P < 0.001). Similarly, callus formation at fracture sites in CNT was significantly increased versus fracture only (P < 0,01). The CNT model mimics a variety of clinically relevant features known from human multiple injury, including TBI, and offers novel approaches for investigation of interactive mechanisms and therapeutic approaches.

Transplanted neural stem cells survive, differentiate, and improve neurological motor function after experimental traumatic brain injury.

OBJECTIVE: Using the neural stem cell (NSC) clone C17.2, we evaluated the ability of transplanted murine NSCs to attenuate cognitive and neurological motor deficits after traumatic brain injury. METHODS: Nonimmunosuppressed C57BL/6 mice (n = 65) were anesthetized and subjected to lateral controlled cortical impact brain injury (n = 52) or surgery without injury (sham operation group, n = 13). At 3 days postinjury, all brain-injured animals were reanesthetized and randomized to receive stereotactic injection of NSCs or control cells (human embryonic kidney cells) into the cortex-hippocampus interface in either the ipsilateral or the contralateral hemisphere. One group of animals (n = 7) was killed at either 1 or 3 weeks postinjury to assess NSC survival in the acute posttraumatic period. Motor function was evaluated at weekly intervals for 12 weeks in the remaining animals, and cognitive (i.e., learning) deficits were assessed at 3 and 12 weeks after transplantation. RESULTS: Brain-injured animals that received either ipsilateral or contralateral NSC transplants showed significantly improved motor function in selected tests as compared with human embryonic kidney cell-transplanted animals during the 12-week observation period. Cognitive dysfunction was unaffected by transplantation at either 3 or 12 weeks postinjury. Histological analyses showed that NSCs survive for as long as 13 weeks after transplantation and were detected in the hippocampus and/or cortical areas adjacent to the injury cavity. At 13 weeks, the NSCs transplanted ipsilateral to the impact site expressed neuronal (NeuN) or astrocytic (glial fibrillary acidic protein) markers but not markers of oligodendrocytes (2'3'cyclic nucleotide 3'-phosphodiesterase), whereas the contralaterally transplanted NSCs expressed neuronal but not glial markers (double-labeled immunofluorescence and confocal microscopy). CONCLUSION: These data suggest that transplanted NSCs can survive in the traumatically injured brain, differentiate into neurons and/or glia, and attenuate motor dysfunction after traumatic brain injury.

Impurity of stem cell graft by murine embryonic fibroblasts - implications for cell-based therapy of the central nervous system.

Stem cells have been demonstrated to possess a therapeutic potential in experimental models of various central nervous system disorders, including stroke. The types of implanted cells appear to play a crucial role. Previously, groups of the stem cell network NRW implemented a feeder-based cell line within the scope of their projects, examining the implantation of stem cells after ischemic stroke and traumatic brain injury. Retrospective evaluation indicated the presence of spindle-shaped cells in several grafts implanted in injured animals, which indicated potential contamination by co-cultured feeder cells (murine embryonic fibroblasts - MEFs). Because feeder-based cell lines have been previously exposed to a justified criticism with regard to contamination by animal glycans, we aimed to evaluate the effects of stem cell/MEF co-transplantation. MEFs accounted for 5.3 ± 2.8% of all cells in the primary FACS-evaluated co-culture. Depending on the culture conditions and subsequent purification procedure, the MEF-fraction ranged from 0.9 to 9.9% of the cell suspensions in vitro. MEF survival and related formation of extracellular substances in vivo were observed after implantation into the uninjured rat brain. Impurity of the stem cell graft by MEFs interferes with translational strategies, which represents a threat to the potential recipient and may affect the graft microenvironment. The implications of these findings are critically discussed.

Pitfalls and fallacies interfering with correct identification of embryonic stem cells implanted into the brain after experimental traumatic injury.

Cell-therapy was proposed to be a promising tool in case of death or impairment of specific cell types. Correct identification of implanted cells became crucial when evaluating the success of transplantation therapy. Various methods of cell labeling have been employed in previously published studies. The use of intrinsic signaling of green fluorescent protein (GFP) has led to a well known controversy in the field of cardiovascular research. We encountered similar methodological pitfalls after transplantation of GFP-transfected embryonic stem cells into rat brains following traumatic brain injury (TBI). As the identification of implanted graft by intrinsic autofluorescence failed, anti-GFP labeling coupled to fluorescent and conventional antibodies was needed to visualize the implanted cells. Furthermore, different cell types with strong intrinsic autofluorescence were found at the sites of injury and transplantation, thus mimicking the implanted stem cells. GFP-positive stem cells were correctly localized, using advanced histological techniques. The activation of microglia/macrophages, accompanying the transplantation post TBI, was shown to be a significant source of artefacts, interfering with correct identification of implanted stem cells. Dependent on the strategy of stem cell tracking, the phagocytosis of implanted cells as observed in this study, might also impede the interpretation of results. Critical appraisal of previously published data as well as a review of different histological techniques provide tools for a more accurate identification of transplanted stem cells.

"The good into the pot, the bad into the crop!"--a new technology to free stem cells from feeder cells.

A variety of embryonic and adult stem cell lines require an initial co-culturing with feeder cells for non-differentiated growth, self renewal and maintenance of pluripotency. However for many downstream ES cell applications the feeder cells have to be considered contaminations that might interfere not just with the analysis of experimental data but also with clinical application and tissue engineering approaches. Here we introduce a novel technique that allows for the selection of pure feeder-freed stem cells, following stem cell proliferation on feeder cell layers. Complete and reproducible separation of feeder and embryonic stem cells was accomplished by adaptation of an automated cell selection system that resulted in the aspiration of distinct cell colonies or fraction of colonies according to predefined physical parameters. Analyzing neuronal differentiation we demonstrated feeder-freed stem cells to exhibit differentiation potentials comparable to embryonic stem cells differentiated under standard conditions. However, embryoid body growth as well as differentiation of stem cells into cardiomyocytes was significantly enhanced in feeder-freed cells, indicating a feeder cell dependent modulation of lineage differentiation during early embryoid body development. These findings underline the necessity to separate stem and feeder cells before the initiation of in vitro differentiation. The complete separation of stem and feeder cells by this new technology results in pure stem cell populations for translational approaches. Furthermore, a more detailed analysis of the effect of feeder cells on stem cell differentiation is now possible, that might facilitate the identification and development of new optimized human or genetically modified feeder cell lines.

Embryonic stem cells produce neurotrophins in response to cerebral tissue extract: Cell line-dependent differences.

In the present study, we compare the capacity of two different embryonic stem (ES) cell lines to secrete neurotrophins in response to cerebral tissue extract derived from healthy or injured rat brains. The intrinsic capacity of the embryonic cell lines BAC7 (feeder cell-dependent cultivation) to release brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3) exceeded the release of these factors by CGR8 cells (feeder cell-free growth) by factors of 10 and 4, respectively. Nerve growth factor (NGF) was secreted only by BAC7 cells. Conditioning of cell lines with cerebral tissue extract derived from healthy or fluid percussion-injured rat brains resulted in a significant time-dependent increase in BDNF release in both cell lines. The increase in BDNF release by BAC7 cells was more pronounced when cells were incubated with brain extract derived from injured brain. However, differences in neurotrophin release associated with the origin of brain extract were at no time statistically significant. Neutrophin-3 and NGF release was inhibited when cell lines were exposed to cerebral tissue extract. The magnitude of the response to cerebral tissue extract was dependent on the intrinsic capacity of the cell lines to release neurotrophins. Our results clearly demonstrate significant variations in the intrinsic capability of different stem cell lines to produce neurotrophic factors. Furthermore, a significant modulation of neurotrophic factor release was observed following conditioning of cell lines with tissue extract derived from rat brains. A significant modulation of neurotrophin release dependent on the source of cerebral tissue extract used was not observed.