Our child's TBI: a rehabilitation engineer's personal experience, technological approach, and lessons learned.

I (JS) am currently a faculty member at The University of Texas at Austin in Mechanical Engineering. My primary research focus is rehabilitation engineering. In May 2020, a week before her fourth birthday, our daughter suffered a severe traumatic brain injury in the early days of the coronavirus pandemic. The purpose of this article is to describe the current state of pediatric neurorehabilitation from technologically-adept parents' first-person perspectives in order to inform and motivate rehabilitation engineering researchers. We describe the medical and personal challenges faced during the aftermath of the accident, the technological approaches to her recovery that my wife (LKS) and I have examined, some of which may be considered beyond standard practice, and the lessons we have absorbed during this period regarding both the state of rehabilitation research and the clinical uptake of rehabilitation technologies. We introduce a set of questions for designers to consider as they create and evaluate new technologies for pediatric rehabilitation.

Inhibition of osteoclast differentiation and bone resorption by N-methylpyrrolidone.

Regulation of RANKL (receptor activator of nuclear factor κB ligand)-induced osteoclast differentiation is of current interest in the development of antiresorptive agents. Osteoclasts are multinucleated cells that play a crucial role in bone resorption. In this study, we investigated the effects of N-methylpyrrolidone (NMP) on the regulation of RANKL-induced osteoclastogenesis. NMP inhibited RANKL-induced tartrate-resistant acid phosphatase activity and the formation of tartrate-resistant acid phosphatase-positive multinucleated cells. The RANKL-induced expression of NFATc1 (nuclear factor of activated T cells, cytoplasmic 1) and c-Fos, which are key transcription factors for osteoclastogenesis, was also reduced by treatment with NMP. Furthermore, NMP induced disruption of the actin rings and decreased the mRNAs of cathepsin K and MMP-9 (matrix metalloproteinase-9), both involved in bone resorption. Taken together, these results suggest that NMP inhibits osteoclast differentiation and attenuates bone resorption. Therefore, NMP could prove useful for the treatment of osteoporosis or other bone diseases associated with excessive bone resorption.

Protein polymer MRI contrast agents: Longitudinal analysis of biomaterials in vivo.

Despite recent advances in tissue engineering to regenerate biological function by combining cells with material supports, development is hindered by inadequate techniques for characterizing biomaterials in vivo. Magnetic resonance imaging is a tomographic technique with high temporal and spatial resolution and represents an excellent imaging modality for longitudinal noninvasive assessment of biomaterials in vivo. To distinguish biomaterials from surrounding tissues for magnetic resonance imaging, protein polymer contrast agents were developed and incorporated into hydrogels. In vitro and in vivo images of protein polymer hydrogels, with and without covalently incorporated protein polymer contrast agents, were acquired by magnetic resonance imaging. T(1) values of the labeled gels were consistently lower when protein polymer contrast agents were included. As a result, the protein polymer contrast agent hydrogels facilitated fate tracking, quantification of degradation, and detection of immune response in vivo. For the duration of the in vivo study, the protein polymer contrast agent-containing hydrogels could be distinguished from adjacent tissues and from the foreign body response surrounding the gels. The hydrogels containing protein polymer contrast agent have a contrast-to-noise ratio 2-fold greater than hydrogels without protein polymer contrast agent. In the absence of the protein polymer contrast agent, hydrogels cannot be distinguished by the end of the gel lifetime.

Multivalent protein polymer MRI contrast agents: controlling relaxivity via modulation of amino acid sequence.

Magnetic resonance imaging is a noninvasive imaging modality with high spatial and temporal resolution. Contrast agents (CAs) are frequently used to increase the contrast between tissues of interest. To increase the effectiveness of MR agents, small molecule CAs have been attached to macromolecules. We have created a family of biodegradable, macromolecular CAs based on protein polymers, allowing control over the CA properties. The protein polymers are monodisperse, random coil, and contain evenly spaced lysines that serve as reactive sites for Gd(III) chelates. The exact sequence and length of the protein can be specified, enabling controlled variation in lysine spacing and molecular weight. Relaxivity could be modulated by changing protein polymer length and lysine spacing. Relaxivities of up to approximately 14 mM(-1) s(-1) per Gd(III) and approximately 461 mM(-1) s(-1) per conjugate were observed. These CAs are biodegradable by incubation with plasmin, such that they can be easily excreted after use. They do not reduce cell viability, a prerequisite for future in vivo studies. The protein polymer CAs can be customized for different clinical diagnostic applications, including biomaterial tracking, as a balanced agent with high relaxivity and appropriate molar mass.

Synthesis and characterization of a new class of cationic protein polymers for multivalent display and biomaterial applications.

Monodisperse protein polymers engineered by biosynthetic techniques are well suited to serve as a basis for creating comb-like polymer architectures for biomaterial applications. We have developed a new class of linear, cationic, random-coil protein polymers designed to act as scaffolds for multivalent display. These polymers contain evenly spaced lysine residues that allow for chemical or enzymatic conjugation of pendant functional groups. Circular dichroism spectroscopy and turbidity experiments have confirmed that these proteins have a random coil structure and are soluble up to at least 65 degrees C. Cell viability assays suggest these constructs are nontoxic in solution up to a concentration of 100 microM. We have successfully attached a small bioactive peptide, a peptoid-peptide hybrid, a poly(ethylene glycol) polymer, and a fluorophore to the protein polymers by chemical or enzymatic coupling, demonstrating their suitability to serve as multivalent scaffolds in solutions or as gels.

Comparative study of NMP-preloaded and dip-loaded membranes for guided bone regeneration of rabbit cranial defects.

Guided bone regeneration (GBR) has been utilized for several decades for the healing of cranio-maxillofacial bone defects and, particularly in the dental field, by creating space with a barrier membrane to exclude soft tissue and encourage bone growth in the membrane-protected volume. Although the first membranes were non-resorbable, a new generation of GBR membranes aims to biodegrade and provide bioactivity for better overall results. The Inion GTR™ poly(lactide-co-glycolide) (PLGA) membrane is not only resorbable but also bioactive, since it includes N-methylpyrrolidone (NMP), which has been shown to promote bone regeneration. In this study, the effects of loading different amounts of NMP onto the membrane through chemical vapour deposition or dipping have been explored. In vitro release demonstrated that lower levels of NMP led to lower NMP concentrations and slower release, based on total NMP loaded in the membrane. The dipped membrane released almost all of the NMP within 15 min, leading to a high NMP concentration. For the in vivo studies in rabbits, 6 mm calvarial defects were created and left untreated or covered with an ePTFE membrane or PLGA membranes dipped in, or preloaded with, NMP. Evaluation of the bony regeneration revealed that the barrier membranes improved bony healing and that a decrease in NMP content improved the performance. Overall, we have demonstrated the potential of these PLGA membranes with a more favourable NMP release profile and the significance of exploring the effect of NMP on these PLGA membranes with regard to bone ingrowth. Copyright © 2014 John Wiley & Sons, Ltd.

Heterodimeric BMP-2/7 for nucleus pulposus regeneration-In vitro and ex vivo studies.

Intervertebral disc (IVD) degeneration is the leading trigger of low back pain, which causes disability and leads to enormous healthcare toll worldwide. Biological treatment with growth factors has evolved as potential therapy for IVD regeneration. Bone morphogenetic protein 2 (BMP-2) and BMP-7 have shown promise in this regard. In the current study, we evaluated the effect of BMP-2/7 heterodimer for disc regeneration both in vitro and in organ culture. Nucleus pulposus (NP) cells isolated from bovine caudal disc were cultured in a fibrin-hyaluronan (FBG-HA) hydrogel for up to 14 days. BMP-2/7 heterodimer covalently incorporated within the hydrogel up-regulated the aggrecan and type II collagen gene expression, and glycosaminoglycan synthesis of NP cells. The activity of the BMP-2/7 heterodimer was dose dependent. The higher dose of BMP-2/7 was further assessed in an IVD whole organ system. After 14 days of culture with cyclic dynamic load, the BMP-2/7 heterodimer delivered into the nucleotomized region showed potential to stimulate the gene expression and synthesis of proteoglycan in the remaining NP tissue after partial nucleotomy. The gene expression level of type I collagen and alkaline phosphatase in the native disc tissue were not affected by BMP-2/7 treatment, indicating no adverse fibroblastic or osteogenic effect on the disc tissue. Intradiscal delivery of BMP-2/7 heterodimer may be a promising therapeutic approach for NP regeneration. The current IVD whole organ partial nucleotomy model may be utilized for screening of other biomaterials or drugs to treat early degenerative disc disorders. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:51-60, 2017.

Fibrin Hydrogel Based Bone Substitute Tethered with BMP-2 and BMP-2/7 Heterodimers.

Current clinically used delivery methods for bone morphogenetic proteins (BMPs) are collagen based and require large concentrations that can lead to dangerous side effects. Fibrin hydrogels can serve as osteoinductive bone substitute materials in non-load bearing bone defects in combination with BMPs. Two strategies to even further optimize such a fibrin based system include employing more potent BMP heterodimers and engineering growth factors that can be covalently tethered to and slowly released from a fibrin matrix. Here we present an engineered BMP-2/BMP-7 heterodimer where an N-terminal transglutaminase substrate domain in the BMP-2 portion provides covalent attachment to fibrin together with a central plasmin substrate domain, a cleavage site for local release of the attached BMP-2/BMP-7 heterodimer under the influence of cell-activated plasmin. In vitro and in vivo results revealed that the engineered BMP-2/BMP-7 heterodimer induces significantly more alkaline phosphatase activity in pluripotent cells and bone formation in a rat calvarial model than the engineered BMP-2 homodimer. Therefore, the engineered BMP-2/BMP-7 heterodimer could be used to reduce the amount of BMP needed for clinical effect.

BMP-2 and BMP-2/7 Heterodimers Conjugated to a Fibrin/Hyaluronic Acid Hydrogel in a Large Animal Model of Mild Intervertebral Disc Degeneration.

Intervertebral disc (IVD) degeneration is etiologically associated with low back pain and is currently only treated in severe cases with spinal fusion. Regenerative medicine attempts to restore degenerated tissue by means of cells, hydrogels, and/or growth factors and can therefore be used to slow, halt, or reverse the degeneration of the IVD in a minimally invasive manner. Previously, the growth factors bone morphogenetic proteins 2 and 7 (BMP-2, -7) were shown to enhance disc regeneration, in vitro and in vivo. Since BMPs have only a short in vivo half-life, and to prevent heterotopic ossification, we evaluated the use of a slow release system for BMP-2 homodimers and BMP-2/7 heterodimers for IVD regeneration. BMP growth factors were conjugated to a fibrin/hyaluronic acid (FB/HA) hydrogel and intradiscally injected in a goat model of mild IVD degeneration to study safety and efficacy. Mild degeneration was induced in five lumbar discs of seven adult Dutch milk goats, by injections with the enzyme chondroitinase ABC. After 12 weeks, discs were treated with either FB/HA-hydrogel only or supplemented with 1 or 5 µg/mL of BMP-2 or BMP-2/7. BMPs were linked to the FB/HA hydrogels using a transglutaminase moiety, to be released through an incorporated plasmin cleavage site. After another 12 weeks, goats were sacrificed and discs were assessed using radiography, MRI T2* mapping, and biochemical and histological analyses. All animals maintained weight throughout the study and no heterotopic bone formation or other adverse effects were noted during follow-up. Radiographs showed significant disc height loss upon induction of mild degeneration. MRI T2* mapping showed strong and significant correlations with biochemistry and histology as shown before. Surprisingly, no differences could be demonstrated in any parameter between intervention groups. To our knowledge, this is the first large animal study evaluating BMPs conjugated to an FB/HA-hydrogel for the treatment of mild IVD degeneration. The conjugated BMP-2 and BMP-2/7 appeared safe, but no disc regeneration was observed. Possible explanations include too low dosages, short follow-up time, and/or insufficient release of the conjugated BMPs. These aspects should be addressed in future studies.

N-methyl pyrrolidone/bone morphogenetic protein-2 double delivery with in situ forming implants.

Bone morphogenetic proteins (BMPs) are growth and differentiation factors involved during development in morphogenesis, organogenesis and later mainly in regeneration processes, in particular in bone where they are responsible for osteoinduction. For more than a decade, recombinant human (rh)BMP-2 has been used in the clinic for lumbar spinal fusion at non-physiological high dosages that appear to be causative for side effects, like male sterility. A possible strategy to reduce the effective amount of rhBMP-2 in the clinic is the co-delivery with an enhancer of BMPs' activity. In an earlier study, we showed that N-methylpyrrolidone (NMP) enhances BMP activity in vitro and in vivo. Here we report on the development of a slow and sustained double delivery of rhBMP-2 and NMP via an in situ forming implant based on poly(lactide-co-glycolide). The results showed that the release of NMP can be adjusted by varying the lactide/glycolide ratio and the polymer's molecular weight. The same applied to rhBMP-2, with release rates that could be sustained from two to three weeks. In the in vivo model of a critical size defect in the calvarial bone of rabbits, the implant containing 50mol% lactide performed better than the one having 75mol% lactide in terms of defect bridging and extent of bony regenerated area. In situ forming implants for the double delivery of the BMP enhancer NMP and rhBMP-2 appear to be promising delivery systems in bone regeneration.

The incorporation of extracellular matrix proteins in protein polymer hydrogels to improve encapsulated beta-cell function.

Biomaterial encapsulation of islets has been proposed to improve the long-term success of islet transplantation by recreating a suitable microenvironment and enhancing cell-matrix interactions that affect cellular function. Protein polymer hydrogels previously showed promise as a biocompatible scaffold by maintaining high cell viability. Here, enzymatically-crosslinked protein polymers were used to investigate the effects of varying scaffold properties and of introducing ECM proteins on the viability and function of encapsulated MIN6 ß-cells. Chemical and mechanical properties of the hydrogel were modified by altering the protein concentrations while collagen IV, fibronectin, and laminin were incorporated to reestablish cell-matrix interactions lost during cell isolation. Rheology indicated all hydrogels formed quickly, resulting in robust, elastic hydrogels with Young's moduli similar to soft tissue. All hydrogels tested supported both high MIN6 ß-cell viability and function and have the potential to serve as an encapsulation platform for islet cell delivery in vivo.

Protein polymer hydrogels: effects of endotoxin on biocompatibility.

Protein polymer-based hydrogels have shown potential for tissue engineering applications, but require biocompatibility testing for in vivo use. Enzymatically crosslinked protein polymer-based hydrogels were tested in vitro and in vivo to evaluate their biocompatibility. Endotoxins present in the hydrogel were removed by Trition X-114 phase separation. The reduction of endotoxins decreased TNF-α production by a macrophage cell line in vitro; however, significant inflammatory response was still present compared to collagen control gels. A branched PEG molecule and dexamethasone were added to the hydrogel to reduce the response. In vitro testing showed a decrease in the TNF-α levels with the addition of dexamethasone. In vivo implantations into the epididymal fat pad of C57/BL6 mice, however, indicated a decreased inflammatory mediated immune response with a hydrogel treated with both PEGylation and endotoxin reduction. This study demonstrates the importance of endotoxin testing and removal in determining the biocompatibility of biomaterials.