Low-frequency ultrasound-mediated cytokine transfection enhances T cell recruitment at local and distant tumor sites.

Robust cytotoxic T cell infiltration has proven to be difficult to achieve in solid tumors. We set out to develop a flexible protocol to efficiently transfect tumor and stromal cells to produce immune-activating cytokines, and thus enhance T cell infiltration while debulking tumor mass. By combining ultrasound with tumor-targeted microbubbles, membrane pores are created and facilitate a controllable and local transfection. Here, we applied a substantially lower transmission frequency (250 kHz) than applied previously. The resulting microbubble oscillation was significantly enhanced, reaching an effective expansion ratio of 35 for a peak negative pressure of 500 kPa in vitro. Combining low-frequency ultrasound with tumor-targeted microbubbles and a DNA plasmid construct, 20% of tumor cells remained viable, and ∼20% of these remaining cells were transfected with a reporter gene both in vitro and in vivo. The majority of cells transfected in vivo were mucin 1+/CD45- tumor cells. Tumor and stromal cells were then transfected with plasmid DNA encoding IFN-ß, producing 150 pg/106 cells in vitro, a 150-fold increase compared to no-ultrasound or no-plasmid controls and a 50-fold increase compared to treatment with targeted microbubbles and ultrasound (without IFN-ß). This enhancement in secretion exceeds previously reported fourfold to fivefold increases with other in vitro treatments. Combined with intraperitoneal administration of checkpoint inhibition, a single application of IFN-ß plasmid transfection reduced tumor growth in vivo and recruited efficacious immune cells at both the local and distant tumor sites.

Ultrasound-Mediated Gene Delivery Enhances Tendon Allograft Integration in Mini-Pig Ligament Reconstruction.

Ligament injuries occur frequently, substantially hindering routine daily activities and sports participation in patients. Surgical reconstruction using autogenous or allogeneic tissues is the gold standard treatment for ligament injuries. Although surgeons routinely perform ligament reconstructions, the integrity of these reconstructions largely depends on adequate biological healing of the interface between the ligament graft and the bone. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would lead to significantly improved ligament graft integration. To test this hypothesis, an anterior cruciate ligament reconstruction procedure was performed in Yucatan mini-pigs. A collagen scaffold was implanted in the reconstruction sites to facilitate recruitment of endogenous mesenchymal stem cells. Ultrasound-mediated reporter gene delivery successfully transfected 40% of cells recruited to the reconstruction sites. When BMP-6 encoding DNA was delivered, BMP-6 expression in the reconstruction sites was significantly enhanced. Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to significantly enhanced osteointegration in all animals 8 weeks after surgery. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively improve ligament reconstruction in large animals, thereby addressing a major unmet orthopedic need and offering new possibilities for translation to the clinical setting.

Recent Advances and Future of Gene Therapy for Bone Regeneration.

PURPOSE OF REVIEW: The purpose of this review is to discuss the recent advances in gene therapy as a treatment for bone regeneration. While most fractures heal spontaneously, patients who present with fracture nonunion suffer from prolonged pain, disability, and often require additional operations to regain musculoskeletal function. RECENT FINDINGS: In the last few years, BMP gene delivery by means of electroporation and sonoporation resulted in repair of nonunion bone defects in mice, rats, and minipigs. Ex vivo transfection of porcine mesenchymal stem cells (MSCs) resulted in bone regeneration following implantation in vertebral defects of minipigs. Sustained release of VEGF gene from a collagen-hydroxyapatite scaffold to the mandible of a human patient was shown to be safe and osteoinductive. In conclusion, gene therapy methods for bone regeneration are systematically becoming more efficient and show proof-of-concept in clinically relevant animal models. Yet, on the pathway to clinical use, more investigation is needed to determine the safety aspects of the various techniques in terms of biodistribution, toxicity, and tumorigenicity.

PTH Induces Systemically Administered Mesenchymal Stem Cells to Migrate to and Regenerate Spine Injuries.

Osteoporosis affects more than 200 million people worldwide leading to more than 2 million fractures in the United States alone. Unfortunately, surgical treatment is limited in patients with low bone mass. Parathyroid hormone (PTH) was shown to induce fracture repair in animals by activating mesenchymal stem cells (MSCs). However, it would be less effective in patients with fewer and/or dysfunctional MSCs due to aging and comorbidities. To address this, we evaluated the efficacy of combination i.v. MSC and PTH therapy versus monotherapy and untreated controls, in a rat model of osteoporotic vertebral bone defects. The results demonstrated that combination therapy significantly increased new bone formation versus monotherapies and no treatment by 2 weeks (P < 0.05). Mechanistically, we found that PTH significantly enhanced MSC migration to the lumbar region, where the MSCs differentiated into bone-forming cells. Finally, we used allogeneic porcine MSCs and observed similar findings in a clinically relevant minipig model of vertebral defects. Collectively, these results demonstrate that in addition to its anabolic effects, PTH functions as an adjuvant to i.v. MSC therapy by enhancing migration to heal bone loss. This systemic approach could be attractive for various fragility fractures, especially using allogeneic cells that do not require invasive tissue harvest.

Quantitative chemical exchange saturation transfer MRI of intervertebral disc in a porcine model.

PURPOSE: Previous studies have associated low pH in intervertebral discs (IVDs) with discogenic back pain. The purpose of this study was to determine whether quantitative CEST (qCEST) MRI can be used to detect pH changes in IVDs in vivo. METHODS: The exchange rate ksw between glycosaminoglycan (GAG) protons and water protons was determined from qCEST analysis. Its dependence on pH value was investigated in GAG phantoms with varying pH and concentrations. The relationship between ksw and pH was studied further in vivo in a porcine model on a 3T MR scanner and validated using a pH meter. Sodium lactate was injected into the IVDs to induce various pH values within the discs ranging from 5 to 7. RESULTS: Phantom and animal results revealed that ksw measured using qCEST MRI is highly correlated with pH level. In the animal studies, the relationship can be described as ksw =9.2 × 106 × 10-pH + 196.9, R2 = 0.7883. CONCLUSION: The exchange rate between GAG and water protons determined from qCEST MRI is closely correlated with pH value. This technique has the potential to noninvasively measure pH in the IVDs of patients with discogenic pain. Magn Reson Med 76:1677-1683, 2016. © 2016 International Society for Magnetic Resonance in Medicine.

Detection of low back pain using pH level-dependent imaging of the intervertebral disc using the ratio of R1ρ dispersion and -OH chemical exchange saturation transfer (RROC).

PURPOSE: Low pH is associated with intervertebral disc (IVD)-generated low back pain (LBP). The purpose of this work was to develop an in vivo pH level-dependent magnetic resonance imaging (MRI) method for detecting discogenic LBP, without using exogenous contrast agents. METHODS: The ratio of R1ρ dispersion and chemical exchange saturation transfer (CEST) (RROC) was used for pH-level dependent imaging of the IVD while eliminating the effect of labile proton concentration. The technique was validated by numerical simulations and studies on phantoms and ex vivo porcine spines. Four male (ages 42.8 ± 18.3) and two female patients (ages 55.5 ± 2.1) with LBP and scheduled for discography were examined with the method on a 3.0 Tesla MR scanner. RROC measurements were compared with discography outcomes using paired t-test. RESULTS: Simulation and phantom results indicated RROC is a concentration independent and pH level-dependent technique. Porcine spine study results found higher RROC value was related to lower pH level. Painful discs based on discography had significant higher RROC values than those with negative diagnosis (P < 0.05). CONCLUSION: RROC imaging is a promising pH level dependent MRI technique that has the potential to be a noninvasive imaging tool to detect painful IVDs in vivo.

Stem cell therapy for osteoporosis.

Osteoporosis is a debilitating disease that affects millions of people worldwide. Current osteoporosis treatments are predominantly bone-resorbing drugs that are associated with several side effects. The use of stem cells for tissue regeneration has raised great hope in various fields of medicine, including musculoskeletal disorders. Stem cell therapy for osteoporosis could potentially reduce the susceptibility of fractures and augment lost mineral density by either increasing the numbers or restoring the function of resident stem cells that can proliferate and differentiate into bone-forming cells. Such osteoporosis therapies can be carried out by exogenous introduction of mesenchymal stem cells (MSCs), typically procured from bone marrow, adipose, and umbilical cord blood tissues or through treatments with drugs or small molecules that recruit endogenous stem cells to osteoporotic sites. The main hurdle with cell-based osteoporosis therapy is the uncertainty of stem cell fate and biodistribution following cell transplantation. Therefore, future advancements will focus on long-term engraftment and differentiation of stem cells at desired bone sites for tangible clinical outcome.

Reliable chemical exchange saturation transfer imaging of human lumbar intervertebral discs using reduced-field-of-view turbo spin echo at 3.0 T.

The reduced field-of-view (rFOV) turbo-spin-echo (TSE) technique, which effectively suppresses bowel movement artifacts, is developed for the purpose of chemical exchange saturation transfer (CEST) imaging of the intervertebral disc (IVD) in vivo. Attempts to quantify IVD CEST signals in a clinical setting require high reliability and accuracy, which is often compromised in the conventionally used technique. The proposed rFOV TSE CEST method demonstrated significantly superior reproducibility when compared with the conventional technique on healthy volunteers, implying it is a more reliable measurement. Phantom study revealed a linear relation between CEST signal and glycosaminoglycan (GAG) concentration. The feasibility of detecting IVD degeneration was demonstrated on a healthy volunteer, indicating that the proposed method is a promising tool to quantify disc degeneration.

PTH promotes allograft integration in a calvarial bone defect.

Allografts may be useful in craniofacial bone repair, although they often fail to integrate with the host bone. We hypothesized that intermittent administration of parathyroid hormone (PTH) would enhance mesenchymal stem cell recruitment and differentiation, resulting in allograft osseointegration in cranial membranous bones. Calvarial bone defects were created in transgenic mice, in which luciferase is expressed under the control of the osteocalcin promoter. The mice were given implants of allografts with or without daily PTH treatment. Bioluminescence imaging (BLI) was performed to monitor host osteprogenitor differentiation at the implantation site. Bone formation was evaluated with the aid of fluorescence imaging (FLI) and microcomputed tomography (µCT) as well as histological analyses. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate the expression of key osteogenic and angiogenic genes. Osteoprogenitor differentiation, as detected by BLI, in mice treated with an allograft implant and PTH was over 2-fold higher than those in mice treated with an allograft implant without PTH. FLI also demonstrated that the bone mineralization process in PTH-treated allografts was significantly higher than that in untreated allografts. The µCT scans revealed a significant increase in bone formation in allograft + PTH treated mice comparing to allograft + PBS treated mice. The osteogenic genes osteocalcin (Oc/Bglap) and integrin binding sialoprotein (Ibsp) were upregulated in the allograft + PTH treated animals. In summary, PTH treatment enhances osteoprogenitor differentiation and augments bone formation around structural allografts. The precise mechanism is not clear, but we show that infiltration pattern of mast cells, associated with the formation of fibrotic tissue, in the defect site is significantly affected by the PTH treatment.

Targeted gene-and-host progenitor cell therapy for nonunion bone fracture repair.

Nonunion fractures present a challenge to orthopedics with no optimal solution. In-vivo DNA electroporation is a gene-delivery technique that can potentially accelerate regenerative processes. We hypothesized that in vivo electroporation of an osteogenic gene in a nonunion radius bone defect site would induce fracture repair. Nonunion fracture was created in the radii of C3H/HeN mice, into which a collagen sponge was placed. To allow for recruitment of host progenitor cells (HPCs) into the implanted sponge, the mice were housed for 10 days before electroporation. Mice were electroporated with either bone morphogenetic protein 9 (BMP-9) plasmid, Luciferase plasmid or injected with BMP-9 plasmid but not electroporated. In vivo bioluminescent imaging indicated that gene expression was localized to the defect site. Microcomputed tomography (µCT) and histological analysis of murine radii electroporated with BMP-9 demonstrated bone formation bridging the bone gap, whereas in the control groups the defect remained unbridged. Population of the implanted collagen sponge by HPCs transfected with the injected plasmid following electroporation was noted. Our data indicate that regeneration of nonunion bone defect can be attained by performing in vivo electroporation with an osteogenic gene combined with recruitment of HPCs. This gene therapy approach may pave the way for regeneration of other skeletal tissues.

Applications of Ultrasound-Mediated Gene Delivery in Regenerative Medicine.

Research on the capability of non-viral gene delivery systems to induce tissue regeneration is a continued effort as the current use of viral vectors can present with significant limitations. Despite initially showing lower gene transfection and gene expression efficiencies, non-viral delivery methods continue to be optimized to match that of their viral counterparts. Ultrasound-mediated gene transfer, referred to as sonoporation, occurs by the induction of transient membrane permeabilization and has been found to significantly increase the uptake and expression of DNA in cells across many organ systems. In addition, it offers a more favorable safety profile compared to other non-viral delivery methods. Studies have shown that microbubble-enhanced sonoporation can elicit significant tissue regeneration in both ectopic and disease models, including bone and vascular tissue regeneration. Despite this, no clinical trials on the use of sonoporation for tissue regeneration have been conducted, although current clinical trials using sonoporation for other indications suggest that the method is safe for use in the clinical setting. In this review, we describe the pre-clinical studies conducted thus far on the use of sonoporation for tissue regeneration. Further, the various techniques used to increase the effectiveness and duration of sonoporation-induced gene transfer, as well as the obstacles that may be currently hindering clinical translation, are explored.

Encapsulation of Human-Bone-Marrow-Derived Mesenchymal Stem Cells in Small Alginate Beads Using One-Step Emulsification by Internal Gelation: In Vitro, and In Vivo Evaluation in Degenerate Intervertebral Disc Model.

Cell microencapsulation in gel beads contributes to many biomedical processes and pharmaceutical applications. Small beads (<300 µm) offer distinct advantages, mainly due to improved mass transfer and mechanical strength. Here, we describe, for the first time, the encapsulation of human-bone-marrow-derived mesenchymal stem cells (hBM-MSCs) in small-sized microspheres, using one-step emulsification by internal gelation. Small (127−257 µm) high-mannuronic-alginate microspheres were prepared at high agitation rates (800−1000 rpm), enabling control over the bead size and shape. The average viability of encapsulated hBM-MSCs after 2 weeks was 81 ± 4.3% for the higher agitation rates. hBM-MSC-loaded microspheres seeded within a glycosaminoglycan (GAG) analogue, which was previously proposed as a mechanically equivalent implant for degenerate discs, kept their viability, sphericity, and integrity for at least 6 weeks. A preliminary in vivo study of hBM-MSC-loaded microspheres implanted (via a GAG-analogue hydrogel) in a rat injured intervertebral disc model demonstrated long-lasting viability and biocompatibility for at least 8 weeks post-implantation. The proposed method offers an effective and reproducible way to maintain long-lasting viability in vitro and in vivo. This approach not only utilizes the benefits of a simple, mild, and scalable method, but also allows for the easy control of the bead size and shape by the agitation rate, which, overall, makes it a very attractive platform for regenerative-medicine applications.

A theranostic 3D ultrasound imaging system for high resolution image-guided therapy.

Microbubble contrast agents are a diagnostic tool with broad clinical impact and an increasing number of indications. Many therapeutic applications have also been identified. Yet, technologies for ultrasound guidance of microbubble-mediated therapy are limited. In particular, arrays that are capable of implementing and imaging microbubble-based therapy in three dimensions in real-time are lacking. We propose a system to perform and monitor microbubble-based therapy, capable of volumetric imaging over a large field-of-view. To propel the promise of the theranostic treatment strategies forward, we have designed and tested a unique array and system for 3D ultrasound guidance of microbubble-based therapeutic protocols based on the frequency, temporal and spatial requirements. Methods: Four 256-channel plane wave scanners (Verasonics, Inc, WA, USA) were combined to control a 1024-element planar array with 1.3 and 2.5 MHz therapeutic and imaging transmissions, respectively. A transducer aperture of ~40×15 mm was selected and Field II was applied to evaluate the point spread function. In vitro experiments were performed on commercial and custom phantoms to assess the spatial resolution, image contrast and microbubble-enhanced imaging capabilities. Results: We found that a 2D array configuration with 64 elements separated by λ-pitch in azimuth and 16 elements separated by 1.5λ-pitch in elevation ensured the required flexibility. This design, of 41.6 mm × 16 mm, thus provided both an extended field-of-view, up to 11 cm x 6 cm at 10 cm depth and steering of ±18° in azimuth and ±12° in elevation. At a depth of 16 cm, we achieved a volume imaging rate of 60 Hz, with a contrast ratio and resolution, respectively, of 19 dB, 0.8 mm at 3 cm and 20 dB and 2.1 mm at 12.5 cm. Conclusion: A single 2D array for both imaging and therapeutics, integrated with a 1024 channel scanner can guide microbubble-based therapy in volumetric regions of interest.

PTH-Induced Bone Regeneration and Vascular Modulation Are Both Dependent on Endothelial Signaling.

The use of a bone allograft presents a promising approach for healing nonunion fractures. We have previously reported that parathyroid hormone (PTH) therapy induced allograft integration while modulating angiogenesis at the allograft proximity. Here, we hypothesize that PTH-induced vascular modulation and the osteogenic effect of PTH are both dependent on endothelial PTH receptor-1 (PTHR1) signaling. To evaluate our hypothesis, we used multiple transgenic mouse lines, and their wild-type counterparts as a control. In addition to endothelial-specific PTHR1 knock-out mice, we used mice in which PTHR1 was engineered to be constitutively active in collagen-1α+ osteoblasts, to assess the effect of PTH signaling activation exclusively in osteoprogenitors. To characterize resident cell recruitment and osteogenic activity, mice in which the Luciferase reporter gene is expressed under the Osteocalcin promoter (Oc-Luc) were used. Mice were implanted with calvarial allografts and treated with either PTH or PBS. A micro-computed tomography-based structural analysis indicated that the induction of bone formation by PTH, as observed in wild-type animals, was not maintained when PTHR1 was removed from endothelial cells. Furthermore, the induction of PTH signaling exclusively in osteoblasts resulted in significantly less bone formation compared to systemic PTH treatment, and significantly less osteogenic activity was measured by bioluminescence imaging of the Oc-Luc mice. Deletion of the endothelial PTHR1 significantly decreased the PTH-induced formation of narrow blood vessels, formerly demonstrated in wild-type mice. However, the exclusive activation of PTH signaling in osteoblasts was sufficient to re-establish the observed PTH effect. Collectively, our results show that endothelial PTHR1 signaling plays a key role in PTH-induced osteogenesis and has implications in angiogenesis.

Molecular targets for tendon neoformation.

Tendons and ligaments are unique forms of connective tissue that are considered an integral part of the musculoskeletal system. The ultimate function of tendon is to connect muscles to bones and to conduct the forces generated by muscle contraction into movements of the joints, whereas ligaments connect bone to bone and provide joint stabilization. Unfortunately, the almost acellular and collagen I-rich structure of tendons and ligaments makes them very poorly regenerating tissues. Injured tendons and ligaments are considered a major clinical challenge in orthopedic and sports medicine. This Review discusses the several factors that might serve as molecular targets that upon activation can enhance or lead to tendon neoformation.

Gene-modified adult stem cells regenerate vertebral bone defect in a rat model.

Vertebral compression fractures (VCFs), the most common fragility fractures, account for approximately 700,000 injuries per year. Since open surgery involves morbidity and implant failure in the osteoporotic patient population, a new minimally invasive biological solution to vertebral bone repair is needed. Previously, we showed that adipose-derived stem cells (ASCs) overexpressing a BMP gene are capable of inducing spinal fusion in vivo. We hypothesized that a direct injection of ASCs, designed to transiently overexpress rhBMP6, into a vertebral bone void defect would accelerate bone regeneration. Porcine ASCs were isolated and labeled with lentiviral vectors that encode for the reporter gene luciferase (Luc) under constitutive (ubiquitin) or inductive (osteocalcin) promoters. The ASCs were first labeled with reporter genes and then nucleofected with an rhBMP6-encoding plasmid. Twenty-four hours later, bone void defects were created in the coccygeal vertebrae of nude rats. The ASC-BMP6 cells were suspended in fibrin gel (FG) and injected into the bone void. A control group was injected with FG alone. The regenerative process was monitored in vivo using microCT, and cell survival and differentiation were monitored using tissue specific reporter genes and bioluminescence imaging (BLI). The surgically treated vertebrae were harvested after 12 weeks and subjected to histological and immunohistochemical (against porcine vimentin) analyses. In vivo BLI detected Luc-expressing cells at the implantation site over a 12-week period. Beginning 2 weeks postoperatively, considerable defect repair was observed in the group treated with ASC-BMP6 cells. The rate of bone formation in the stem cell-treated group was two times faster than that in the FG-treated group, and bone volume at the end point was 2-fold compared to the control group. Twelve weeks after cell injection the bone volume within the void reached the volume measured in native vertebrae. Immunostaining against porcine vimentin indicated that the ASC-BMP6 cells contributed to new bone formation. Here we show the potential of injections of BMP-modified ASCs to repair vertebral bone defects in a rat model. Our results could pave the way to a novel approach for the biological treatment of traumatic and osteoporosis-related vertebral bone injuries.

Stem Cells and Exosomes: New Therapies for Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IVDD) occurs as a result of an imbalance of the anabolic and catabolic processes in the intervertebral disc, leading to an alteration in the composition of the extracellular matrix (ECM), loss of nucleus pulposus (NP) cells, excessive oxidative stress and inflammation. Degeneration of the IVD occurs naturally with age, but mechanical trauma, lifestyle factors and certain genetic abnormalities can increase the likelihood of symptomatic disease progression. IVDD, often referred to as degenerative disc disease (DDD), poses an increasingly substantial financial burden due to the aging population and increasing incidence of obesity in the United States. Current treatments for IVDD include pharmacological and surgical interventions, but these lack the ability to stop the progression of disease and restore the functionality of the IVD. Biological therapies have been evaluated but show varying degrees of efficacy in reversing disc degeneration long-term. Stem cell-based therapies have shown promising results in the regeneration of the IVD, but face both biological and ethical limitations. Exosomes play an important role in intercellular communication, and stem cell-derived exosomes have been shown to maintain the therapeutic benefit of their origin cells without the associated risks. This review highlights the current state of research on the use of stem-cell derived exosomes in the treatment of IVDD.

Intradiscal quantitative chemical exchange saturation transfer MRI signal correlates with discogenic pain in human patients.

Low back pain (LBP) is often a result of a degenerative process in the intervertebral disc. The precise origin of discogenic pain is diagnosed by the invasive procedure of provocative discography (PD). Previously, we developed quantitative chemical exchange saturation transfer (qCEST) magnetic resonance imaging (MRI) to detect pH as a biomarker for discogenic pain. Based on these findings we initiated a clinical study with the goal to evaluate the correlation between qCEST values and PD results in LBP patients. Twenty five volunteers with chronic low back pain were subjected to T2-weighted (T2w) and qCEST MRI scans followed by PD. A total of 72 discs were analyzed. The average qCEST signal value of painful discs was significantly higher than non-painful discs (p = 0.012). The ratio between qCEST and normalized T2w was found to be significantly higher in painful discs compared to non-painful discs (p = 0.0022). A receiver operating characteristics (ROC) analysis indicated that qCEST/T2w ratio could be used to differentiate between painful and non-painful discs with 78% sensitivity and 81% specificity. The results of the study suggest that qCEST could be used for the diagnosis of discogenic pain, in conjunction with the commonly used T2w scan.

Monitoring of the effect of intervertebral disc nucleus pulposus ablation by MRI.

In order to investigate intervertebral disc (IVD) degeneration and repair, a quantitative non-invasive tool is needed. Various MRI methods including qCPMG, which yields dipolar echo relaxation time (T(DE)), magnetization transfer contrast (MTC), and (1)H and (2)H double quantum filtered (DQF) MRI were used in the present work to monitor changes in rat IVD after ablation of the nucleus pulposus (NP), serving as a model of severe IVD degeneration. In the intact IVD, a clear distinction between the annulus fibrosus (AF) and the NP is obtained on T(2) and T(DE) weighted images as well as on MTC maps, reflecting the high concentration of ordered collagen fibers in the AF. After ablation of the NP, the distinction between the compartments is lost. T(2) and T(DE) relaxation times are short throughout the disc and MTC is high. (1)H and (2)H DQF signal, which in intact discs is obtained only for the AF, is now observable throughout the tissue. These results indicate that after ablation, there is an ingression of collagen fibers from the AF into the area that was previously occupied by the NP, as was confirmed by histology.

Peptide-modified "smart" hydrogels and genetically engineered stem cells for skeletal tissue engineering.

Stimuli responsive or "smart" hydrogels are of interest for tissue-engineering applications, featuring the advantages of minimally invasive application. Currently, these materials have yet to be used as a biological replacement in restoring the function of damaged tissues or organs. The aim of this study was to demonstrate the advantages of thermoresponsive, peptide-containing hydrogels as a supportive matrix for genetically engineered stem cells. We used injectable hydrogels, enabling cell delivery to the desired site and providing adequate scaffolding postimplantation. Thermoresponsive hydrogels were developed based on amphiphilic block copolymers of polyethylene-oxide and polypropylene-oxide end-capped with methacrylate or maleimide entities and further reacted with RGD-containing peptides. Cell metabolic activity and survival within those hydrogels was studied, illustrating that the stable peptide-polymer conjugate is required for prolonged cell support. The unique polymer characteristics, combined with its enhanced cell interactions, suggest the potential use of these biomaterials in various tissue engineering applications.