The Effects of Stably Tethered BMP-2 on MC3T3-E1 Preosteoblasts Encapsulated in a PEG Hydrogel.

Bone morphogenetic protein-2 (BMP-2) is a clinically used osteoinductive growth factor. With a short half-life and side effects, alternative delivery approaches are needed. This work examines thiolation of BMP-2 for chemical attachment to a poly(ethylene glycol) hydrogel using thiol-norbornene click chemistry. BMP-2 retained bioactivity post-thiolation and was successfully tethered into the hydrogel. To assess tethered BMP-2 on osteogenesis, MC3T3-E1 preosteoblasts were encapsulated in matrix metalloproteinase (MMP)-sensitive hydrogels containing RGD and either no BMP-2, soluble BMP-2 (5 nM), or tethered BMP-2 (40-200 nM) and cultured in a chemically defined medium containing dexamethasone for 7 days. The hydrogel culture supported MC3T3-E1 osteogenesis regardless of BMP-2 presentation, but tethered BMP-2 augmented the osteogenic response, leading to significant increases in osteomarkers, Bglap and Ibsp. The ratio, Ibsp-to-Dmp1, highlighted differences in the extent of differentiation, revealing that without BMP-2, MC3T3-E1 cells showed a higher expression of Dmp1 (low ratio), but an equivalent expression with tethered BMP-2 and more abundant bone sialoprotein. In addition, this work identified that dexamethasone contributed to Ibsp expression but not Bglap or Dmp1 and confirmed that tethered BMP-2 induced the BMP canonical signaling pathway. This work presents an effective method for the modification and incorporation of BMP-2 into hydrogels to enhance osteogenesis.

Assessment and prevention of cartilage degeneration surrounding a focal chondral defect in the porcine model.

Focal defects in articular cartilage are unable to self-repair and, if left untreated, are a leading risk factor for osteoarthritis. This study examined cartilage degeneration surrounding a defect and then assessed whether infilling the defect prevents degeneration. We created a focal chondral defect in porcine osteochondral explants and cultured them ex vivo with and without dynamic compressive loading to decouple the role of loading. When compared to a defect in a porcine knee four weeks post-injury, this model captured loss in sulfated glycosaminoglycans (sGAGs) along the defect's edge that was observed in vivo, but this loss was not load dependent. Loading, however, reduced the indentation modulus of the surrounding cartilage. After infilling with in situ polymerized hydrogels that were soft (100 kPa) or stiff (1 MPa) and which produced swelling pressures of 13 and 310 kPa, respectively, sGAG loss was reduced. This reduction correlated with increased hydrogel stiffness and swelling pressure, but was not affected by loading. This ex vivo model recapitulates sGAG loss surrounding a defect and, when infilled with a mechanically supportive hydrogel, degeneration is minimized.

Controlled Mechanical Property Gradients Within a Digital Light Processing Printed Hydrogel-Composite Osteochondral Scaffold.

Tissue engineered scaffolds are needed to support physiological loads and emulate the micrometer-scale strain gradients within tissues that guide cell mechanobiological responses. We designed and fabricated micro-truss structures to possess spatially varying geometry and controlled stiffness gradients. Using a custom projection microstereolithography (µSLA) system, using digital light projection (DLP), and photopolymerizable poly(ethylene glycol) diacrylate (PEGDA) hydrogel monomers, three designs with feature sizes < 200 µm were formed: (1) uniform structure with 1 MPa structural modulus ( E ) designed to match equilibrium modulus of healthy articular cartilage, (2) E = 1 MPa gradient structure designed to vary strain with depth, and (3) osteochondral bilayer with distinct cartilage ( E = 1 MPa) and bone ( E = 7 MPa) layers. Finite element models (FEM) guided design and predicted the local mechanical environment. Empty trusses and poly(ethylene glycol) norbornene hydrogel-infilled composite trusses were compressed during X-ray microscopy (XRM) imaging to evaluate regional stiffnesses. Our designs achieved target moduli for cartilage and bone while maintaining 68-81% porosity. Combined XRM imaging and compression of empty and hydrogel-infilled micro-truss structures revealed regional stiffnesses that were accurately predicted by FEM. In the infilling hydrogel, FEM demonstrated the stress-shielding effect of reinforcing structures while predicting strain distributions. Composite scaffolds made from stiff µSLA-printed polymers support physiological load levels and enable controlled mechanical property gradients which may improve in vivo outcomes for osteochondral defect tissue regeneration. Advanced 3D imaging and FE analysis provide insights into the local mechanical environment surrounding cells in composite scaffolds.

Osteogenic effects of covalently tethered rhBMP-2 and rhBMP-9 in an MMP-sensitive PEG hydrogel nanocomposite.

While bone morphogenic protein-2 (BMP-2) is one of the most widely studied BMPs in bone tissue engineering, BMP-9 has been purported to be a highly osteogenic BMP. This work investigates the individual osteogenic effects of recombinant human (rh) BMP-2 and rhBMP-9, when tethered into a hydrogel, on encapsulated human mesenchymal stem cells (MSCs). A matrix-metalloproteinase (MMP)-sensitive hydrogel nanocomposite, comprised of poly(ethylene glycol) crosslinked with MMP-sensitive peptides, tethered RGD, and entrapped hydroxyapatite nanoparticles was used. The rhBMPs were functionalized with free thiols and then covalently tethered into the hydrogel by a thiol-norbornene photoclick reaction. rhBMP-2 retained its full bioactivity post-thiolation, while the bioactivity of rhBMP-9 was partially reduced. Nonetheless, both rhBMPs were highly effective at enhancing osteogenesis over 12-weeks in a chemically-defined medium. Expression of ID1 and osterix, early markers of osteogenesis; collagen type I, a main component of the bone extracellular matrix (ECM); and osteopontin, bone sialoprotein II and dentin matrix protein I, mature osteoblast markers, increased with increasing concentrations of tethered rhBMP-2 or rhBMP-9. When comparing the two BMPs, rhBMP-9 led to more rapid collagen deposition and greater mineralization long-term. In summary, rhBMP-2 retained its bioactivity post-thiolation while rhBMP-9 is more susceptible to thiolation. Despite this shortcoming with rhBMP-9, both rhBMPs when tethered into this hydrogel, enhanced osteogenesis of MSCs, leading to a mature osteoblast phenotype surrounded by a mineralized ECM. STATEMENT OF SIGNIFICANCE: Osteoinductive hydrogels are a promising vehicle to deliver mesenchymal stem cells (MSCs) for bone regeneration. This study examines the in vitro osteoinductive capabilities when tethered bone morphogenic proteins (BMPs) are incorporated into a degradable biomimetic hydrogel with cell adhesive ligands, matrix metalloproteinase sensitive crosslinks for cell-mediated degradation, and hydroxyapatite nanoparticles. This study demonstrates that BMP-2 is readily thiolated and tethered without loss of bioactivity while bioactivity of BMP-9 is more susceptible to immobilization. Nonetheless, when either BMP2 or BMP9 are tethered into this hydrogel, osteogenesis of human MSCs is enhanced, bone extracellular matrix is deposited, and a mature osteoblast phenotype is achieved. This bone-biomimetic hydrogel is a promising design for stem cell-mediated bone regeneration.

A 3D printed mimetic composite for the treatment of growth plate injuries in a rabbit model.

Growth plate injuries affecting the pediatric population may cause unwanted bony repair tissue that leads to abnormal bone elongation. Clinical treatment involves bony bar resection and implantation of an interpositional material, but success is limited and the bony bar often reforms. No treatment attempts to regenerate the growth plate cartilage. Herein we develop a 3D printed growth plate mimetic composite as a potential regenerative medicine approach with the goal of preventing limb length discrepancies and inducing cartilage regeneration. A poly(ethylene glycol)-based resin was used with digital light processing to 3D print a mechanical support structure infilled with a soft cartilage-mimetic hydrogel containing chondrogenic cues. Our biomimetic composite has similar mechanical properties to native rabbit growth plate and induced chondrogenic differentiation of rabbit mesenchymal stromal cells in vitro. We evaluated its efficacy as a regenerative interpositional material applied after bony bar resection in a rabbit model of growth plate injury. Radiographic imaging was used to monitor limb length and tibial plateau angle, microcomputed tomography assessed bone morphology, and histology characterized the repair tissue that formed. Our 3D printed growth plate mimetic composite resulted in improved tibial lengthening compared to an untreated control, cartilage-mimetic hydrogel only condition, and a fat graft. However, in vivo the 3D printed growth plate mimetic composite did not show cartilage regeneration within the construct histologically. Nevertheless, this study demonstrates the feasibility of a 3D printed biomimetic composite to improve limb lengthening, a key functional outcome, supporting its further investigation as a treatment for growth plate injuries.

Biomimetic and mechanically supportive 3D printed scaffolds for cartilage and osteochondral tissue engineering using photopolymers and digital light processing.

Successful 3D scaffold designs for musculoskeletal tissue engineering necessitate full consideration of the form and function of the tissues of interest. When designing structures for engineering cartilage and osteochondral tissues, one must reconcile the need to develop a mechanically robust system that maintains the health of cells embedded in the scaffold. In this work, we present an approach that decouples the mechanical and biochemical needs and allows for the independent development of the structural and cellular niches in a scaffold. Using the highly tuned capabilities of digital light processing-based stereolithography, structures with complex architectures are achieved over a range of effective porosities and moduli. The 3D printed structure is infilled with mesenchymal stem cells and soft biomimetic hydrogels, which are specifically formulated with extracellular matrix analogs and tethered growth factors to provide selected biochemical cues for the guided differentiation towards chondrogenesis and osteogenesis. We demonstrate the ability to utilize these structures to (a) infill a focal chondral defect and mitigate macroscopic and cellular level changes in the cartilage surrounding the defect, and (b) support the development of a stratified multi-tissue scaffold for osteochondral tissue engineering.

Development of pedagogical knowledge among learning assistants.

BACKGROUND: Successful outcomes of the Learning Assistant (LA) model include increased learning outcomes in STEM gateway courses and increased persistence to graduation among LAs and the students they serve. While there are many possible reasons that the LA program is effective, the pedagogical development of the LAs themselves has not yet been systematically studied. The research reported here investigated how deeply first-time LAs enrolled in a one-semester pedagogy course took up the language associated with the course's essential pedagogical principles. By reviewing prior research as well as assessing our target population and our pedagogy course learning goals, we developed a set of three essential pedagogical principles that are critical for effective classroom instruction and developed a coding scheme for identifying these principles in LAs' written work. We then looked at LA's development of language with respect to these principles by analyzing weekly teaching reflections submitted by LAs during five iterations of our pedagogy course. RESULTS: Our research indicated that the language used to introduce particular pedagogical principles might play an important role in initiating LAs' uptake of these concepts.We found that LAs began to develop an understanding of the language that values students' prior ideas in learning, but the depth of this understanding varied. In addition, LAs did not demonstrate as much growth in their language with respect to the formative assessment or to the idea that students play a role in constructing knowledge. CONCLUSIONS: In developing a pedagogy course for LAs, relating to their prior backgrounds in STEM appears to be critical. Using language that is accessible seems to increase LAs' ability to develop pedagogical principles. Although LAs' development of language related to the essential pedagogical principles is small, it may be enough to allow them to create contexts that facilitate learning.