Persistent Musculoskeletal Deficits in Pediatric,Adolescent and Young Adult Survivors of Allogeneic Hematopoietic Stem-Cell Transplantation

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a common therapy for pediatric hematologic malignancies. With improved supportive care, addressing treatment-related late effects is at the forefront of survivor long-term health and quality of life. We previously demonstrated that alloHSCT survivors had increased adiposity, decreased lean mass, and lower bone density and strength, 7 years (median) from alloHSCT compared to their healthy peers. Yet it is unknown whether these deficits persist. Our longitudinal study characterized changes in muscle and bone over a period of 3.4 (range, 2.0 to 4.9) years in 47 childhood alloHSCT survivors, age 5–26 years at baseline (34% female). Tibia cortical bone geometry and volumetric density and lower leg muscle cross-sectional area (MCSA) were assessed via peripheral quantitative computed tomography (pQCT). Anthropometric and pQCT measurements were converted to age, sex, and ancestry-specific standard deviation scores, adjusted for leg length. Muscle-specific force was assessed as strength relative to MCSA adjusted for leg length (strength Z-score). Measurements were compared to a healthy reference cohort (n = 921), age 5–30 years (52% female). At baseline and follow-up, alloHSCT survivors demonstrated lower height Z-scores, weight Z-scores, and leg length Z-scores compared to the healthy reference cohort. Deficits in MCSA, trabecular volumetric bone density, and cortical bone size and estimated strength (section modulus) were evident in survivors (all p < 0.05). Between the two study time points, anthropometric, muscle, and bone Z-scores did not change significantly in alloHSCT survivors. Approximately 15% and 17% of alloHSCT survivors had MCSA and section modulus Z-score < −2.0, at baseline and follow-up, respectively. Furthermore, those with a history of total body irradiation compared to those without demonstrated lower MCSA at follow-up. The persistent muscle and bone deficits in pediatric alloHSCT survivors support the need for strategies to improve bone and muscle health in this at-risk population. © 2022 American Society for Bone and Mineral Research (ASBMR).     

PTH‐enhanced structural allograft healing is associated with decreased angiopoietin‐2–mediated arteriogenesis,mast cell accumulation,and fibrosis

Recombinant parathyroid hormone (rPTH) therapy has been evaluated for skeletal repair in animal studies and clinical trials based on its known anabolic effects, but its effects on angiogenesis and fibrosis remain poorly understood. We examined the effects of rPTH therapy on blood vessel formation and osseous integration in a murine femoral allograft model, which caused a significant increase in small vessel numbers, and decreased large vessel formation (p < 0.05). Histology showed that rPTH also reduced fibrosis around the allografts to similar levels observed in live autografts, and decreased mast cells at the graft‐host junction. Similar effects on vasculogenesis and fibrosis were observed in femoral allografts from Col1caPTHR transgenic mice. Gene expression profiling revealed rPTH‐induced angiopoietin‐1 (8‐fold), while decreasing angiopoietin‐2 (70‐fold) at day 7 of allograft healing. Finally, we show anti‐angiopoietin‐2 peptibody (L1‐10) treatment mimics rPTH effects on angiogenesis and fibrosis. Collectively, these findings show that intermittent rPTH treatment enhances structural allograft healing by two processes: (1) anabolic effects on new bone formation via small vessel angiogenesis, and (2) inhibition of angiopoietin‐2–mediated arteriogenesis. The latter effect may function as a vascular sieve to limit mast cell access to the site of tissue repair, which decreases fibrosis around and between the fractured ends of bone. Thus, rPTH therapy may be generalizable to all forms of tissue repair that suffer from limited biointegration and excessive fibrosis. © 2013 American Society for Bone and Mineral Research.     

Adsorption of matrix metalloproteinases onto biomedical polymers: a new aspect in biological acceptance

Matrix metalloproteinases (MMPs) are zinc-dependent enzymes involved in the remodelling of connective tissues during the development and wound healing. Moreover, two MMPs, Gelatinase A (MMP-2) and Gelatinase B (MMP-9), are also present in body fluids such as blood and urine and, therefore, they can be in contact with implanted biomaterials and can be adsorbed onto their surface. In order to test this hypothesis disks of different polymers (polystyrene (PS), polyvinyl chloride (PVC), poly(D,L-lactide) (PLA), polymethyl methacrylate (PMMA) and poly(2-hydroxyethyl methacrylate) (PHEMA)) have been exposed to human plasma and adsorbed proteins have been eluted and analyzed. Using Western blot and substrate zymography analysis, we observed that both MMP-2 and MMP-9 adsorbed onto the surfaces of all the polymers, especially hydrophilic ones (PMMA and PHEMA) and PLA, in both the active and inactive forms. Furthermore, we observed that adhesion of human granulocyte neutophils to PMMA, the polymer that adsorbed the higher quantity of MMP-2 and MMP-9 compared to the others, was reduced by more that 50% by the presence of a gelatinase inhibitor. This data suggest a surprising role of these absorbed enzymes in the adhesion of neutrophil onto some polymeric biomaterials surface and, therefore, in the setting of inflammation.     

Stem-cell culture on patterned bio-functional surfaces

Bio-functional surfaces have been created by printing proteins on antifouling surfaces in a customised geometry. Human umbilical cord neural stem cells incubated on the samples readily attach to the protein defined domains, where they have been monitored during 21 days of culture. The stability of the pattern varies with the density of cells anchored to the microstamped proteins. Highly packed cell patterned domains favoured non-differentiated mode, while low-density areas allowed the spreading out of the cells and differentiation. Tailoring the geometry (pattern size and distances) enables improving the monitoring of the stem cells' developmental processes. The biocompatible surfaces can serve as a model to study processes accompanying stem cell neural lineage commitment.     

Uptake of Enzymatically-Digested Hyaluronan by Liver Endothelial Cells in Vivo and in Vitro

Intravenously-injected hyaluronan (HA) is distributed into liver in which endothelium is a site of uptake and degradation of HA. The role and fate of HA have been widely investigated; however, effects of size and dose of HA on its metabolism have not been well documented yet. To investigate these effects, we prepared fluorescein-labeled HAs, according to the modified methods described by de Belder and Wik, which were enzymatically digested. The 90 kDa fluorescein-labeled HA gradually accumulated in a liver that was distributed into the endothelium; however, 10 kDa or less HA did not. Cell fractionation and flow cytometry further demonstrated the cell of uptake in the liver is an endothelial cell, both in vivo and in vitro. Interestingly, the largest uptake by liver endothelial cells in vitro was observed in 10 kDa HA, even though which did not accumulate in liver in vivo. These results suggest that the result observed with 10 kDa HA in vivo is due to the rapid excretion in urine. Thus, inhibiting of the digestion or suppressing of the urinary excretion would enhance uptake of HA in vivo. These ideas may help to deliver drugs or genes targeting to liver endothelium.     

Lipocalin-2-loaded Amphiphilic Polyanhydride Microparticles Accelerate Cell Migration

This work demonstrates that amphiphilic polyanhydride microparticles based on co-polymers of 1,6-bis(p-carboxyphenoxy)hexane (CPH) and 1,6-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) provide stabilizing environments for proteins. A cryogenic atomization method was used to fabricate protein-loaded polyanhydride microparticles. These microparticles were tested for their ability to provide controlled delivery of lipocalin 2 (Lcn2) and to maintain its structure and function. Lcn2 is an acute-phase protein suspected to play a role in cell migration and tissue repair. The in vitro release kinetics of Lcn2 from the microparticles were a function of the chemistry of the polymer carrier. The biological activity of Lcn2 released from polyanhydride microparticles was investigated by its ability to stimulate migration of human colon epithelial cells (HCT116). Lcn2 released from 50:50 and 20:80 CPTEG/CPH microparticles maintained its biological activity as demonstrated by the increased rate of cell migration. In addition, the Lcn2-loaded 50:50 and 20:80 CPTEG/CPH microparticles promoted cell migration over that of the Lcn2 administered alone. This was interpreted as the ability of the amphiphilic microparticles to stabilize the encapsulated protein and release it in a controlled manner over a period of time. This work demonstrates the potential for therapeutic use of amphiphilic polyanhydride microparticles as protein/drug carriers.     

Patterning Collagen/Poloxamine-Methacrylate Hydrogels for Tissue-Engineering-Inspired Microfluidic and Laser Lithography Applications

The ability to pattern semi-synthetic collagen/poloxamine-methacrylate hydrogels into straight-channel flow circuits and sub-millimeter-sized rectangular blocks for tissue-engineering applications was evaluated. Endothelial cells, grown on the surface of flat collagen/poloxamine-methacrylate hydrogels, proliferated, expressed ICAM-1 (but not VCAM-1) and began to detach after 6 days. Seeding endothelial cells onto the lumen surface of straight collagen/poloxamine-methacrylate flow channels increased ICAM-1 and VCAM-1 expression, and exposure to laminar shear stress (0.3–10 dyn/cm²) was unable to attenuate activation on the relatively few cells that were able to withstand flow associated ablation. The enrichment of poloxamine-methacrylate at the lumen surface during fabrication likely caused the decrease in cell attachment and increased activation. To micropattern more complex structures, confocal microscopy UV laser lithography was used to selectively cross-link a HepG2-containing pre-polymer solution of collagen/poloxamine-methacrylate. Turbidity (caused by suspended cells and the incomplete miscibility of collagen and poloxamine-methacrylate) scattered the UV laser energy and necessitated the optimization of exposure times with respect to cross-linking extent and cell viability. Free radical diffusion beyond the bounds of the initial photopattern reduced the resolution of the structures and created a weakly cross-linked periphery around the original pattern. Over time, HepG2 cells migrated towards the less cross-linked periphery and proliferated, creating a non-uniform distribution of cells.     

Time-Dependent Alginate/Polyvinyl Alcohol Hydrogels as Injectable Cell Carriers

Alginate hydrogels have been widely utilized as cell carriers due to their simplicity for fabricating cell-immobilized gel beads or 3-dimentional porous scaffolds, biocompatibility and non-toxicity to cells. Generally alginate hydrogels have been produced by contacting alginate solution with CaCl₂ as a cross-linking agent. However, the major disadvantages of this system are that the gelation rate is too fast and hard to control, and the prepared alginate gels cannot be injected. Injectable alginates have been prepared by using CaSO₄ or CaCO₃ as a cross-linking agent. However, the gelation rate of alginate with CaCO₃ is slow owing to the low solubility of CaCO₃ in water, while that with CaSO₄ is too fast to form uniform gels. In this study, we prepared injectable alginate/polyvinyl alcohol (PVA) blend hydrogels with controllable gelation rate by using CaSO₄ as a cross-linking agent and Na₂HPO₄ as a cross-linking retardation agent. The gelation rate could be controlled by adjusting CaSO₄/Na₂HPO₄ ratio in the solution. The alginate and PVA showed good compatibility in aqueous solutions or gels. The gelation rate of alginate increased with increasing Na₂HPO₄ and decreasing CaSO₄ concentrations, as expected. The PVA itself in the alginate/PVA blend did not affect the gelation rate. All alginate/PVA hydrogels demonstrated some extraction of PVA, but the extraction extent was not much even after 7 days immersion in water. The alginate/PVA hydrogels were examined for their in vitro cell compatibility by the culture of chondrocytes (human chondrocyte cell line) in the gels up to 28 days. The cells were grown almost linearly in the alginate/PVA hydrogels with higher PVA compositions showing better cell growth. The GAG contents from the cells in the hydrogels did not show dramatic changes with culture time, however, they also increased gradually in the alginate/PVA hydrogels with higher PVA composition. The PVA in the hydrogels seemed to play positive roles for the growth and activity of chondrocytes. The alginate/PVA hydrogels with controllable gelation rate are expected to be applicable as injectable cell carriers.     

Modulating the Activities of Human Mesenchymal Stem Cells (hMSCs) and C3A/HepG2 Hepatoma Cells by Modifying the Surface Characteristics of Poly(3-hydroxybutyrate-co-3-hydroxyhexnoate) (PHBHHx)

The new biodegradable polyester poly(3-hydroxybutyrate-co-3-hydroxyhexnoate) (PHBHHx) has a potential application in tissue engineering. The aim of this study was to present a deeper picture of the relationship between the cellular behavior and the surface characteristics of PHBHHx films. The pristine PHBHHx film was prepared by adopting the compression-molding method, and then the acrylic acid molecules were grafted on PHBHHx membrane surface by UV irradiation. The hydrophilic nature and surface roughness of various PHBHHx films were controlled by adjusting the acrylic acid concentration and the UV irradiation time. Although the surface characteristics of various PHBHHx films could not affect the metabolic activity of hMSCs, the performance of morphology of hMSCs was deeply affected by the hydrophilic nature and the orientation of surface scars. The hydrophilic nature would effectively improve the spread of hMSCs, and the orientation of surface scars would guide the growth direction of cytoskeleton (actin) inside hMSCs. In contrast, the behaviors of C3A/HepG2 hepatoma cells presented an opposite outcomes. Those surface characteristics were obviously associated with the performance of metabolic activity of C3A cells, but not with the morphology of C3A cells. Both hMSCs and C3A cells have unique cellular characteristics; therefore, their responses to environmental stimulations are significantly different.     

Fabrication and Characterization of Hydroxyapatite-Coated Polystyrene Disks for Use in Osteoprogenitor Cell Culture

A simple method is reported for fabricating polystyrene disk inserts coated with biomimetic carbonated hydroxyapatite (cHA) to be used for culturing osteoprogenitor cells or other stem cells. Roughened disks cut from tissue-culture polystyrene (TCPS) were coated in simulated body fluid with 5 × normal physiologic ionic concentrations (SBFx5) by a 2-step, 2-day method. The coatings were rigorously characterized by various methods and assessed in cell culture. An adherent, nearly 10 mm thick, relatively uniform layer of single-phase cHA was formed in two days. MC3T3-E1 and mouse calvaria-derived osteoprogenitor cells (pCOBs) were cultured on the cHA for various time points. Despite less initial attachment of both cell types to the cHA, proliferation rates on cHA were similar to that on TCPS. Two-fold greater cell attachment (P < 0.05) of the MC3T3-E1 cells was observed relative to the pCOBs, on both the TCPS and the cHA. Importantly, the coatings were relatively smooth, without the extensive agglomerates observed in other studies and remained adherent and morphologically unchanged after 21 days of culture. This technique can be used to rapidly produce high-quality cHA-coated TCPS disks for cell-culture studies.