Palovarotene inhibits connective tissue progenitor cell proliferation in a rat model of combat-related heterotopic ossification.

Heterotopic ossification (HO) develops in the extremities of wounded service members and is common in the setting of high-energy penetrating injuries and blast-related amputations. No safe and effective prophylaxis modality has been identified for this patient population. Palovarotene has been shown to reduce bone formation in traumatic and genetic models of HO. The purpose of this study was to determine the effects of Palovarotene on inflammation, progenitor cell proliferation, and gene expression following a blast-related amputation in a rodent model (n = 72 animals), as well as the ability of Raman spectroscopy to detect early HO before radiographic changes are present. Treatment with Palovarotene was found to dampen the systemic inflammatory response including the cytokines IL-6 (p = 0.01), TNF-α (p = 0.001), and IFN-γ (p = 0.03) as well as the local inflammatory response via a 76% reduction in the cellular infiltration at post-operative day (POD)-7 (p = 0.03). Palovarotene decreased osteogenic connective tissue progenitor (CTP-O) colonies by as much as 98% both in vitro (p = 0.04) and in vivo (p = 0.01). Palovarotene treated animals exhibited significantly decreased expression of osteo- and chondrogenic genes by POD-7, including BMP4 (p = 0.02). Finally, Raman spectroscopy was able to detect differences between the two groups by POD-1 (p < 0.001). These results indicate that Palovarotene inhibits traumatic HO formation through multiple inter-related mechanisms including anti-inflammatory, anti-proliferative, and gene expression modulation. Further, that Raman spectroscopy is able to detect markers of early HO formation before it becomes radiographically evident, which could facilitate earlier diagnosis and treatment. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1135-1144, 2018.

Slowing the Onset of Hypoxia Increases Colony Forming Efficiency of Connective Tissue Progenitor Cells In Vitro.

BACKGROUND: Survival and colony formation by transplanted tissue derived connective tissue progenitor cells (CTPs) are thought to be important factors in the success of clinical tissue engineering strategies for bone regeneration. Transplantation of cells into defects larger than a few millimeters expose cells to a profoundly hypoxic environment. This study tested the hypothesis that delaying the onset of hypoxia will improve the survival and performance of CTPs in vitro. METHODS: To mimic declines seen in an avascular in vivo bone defect, colony forming efficiency by marrow derived nucleated cells was assessed under osteogenic conditions. Variation in the rate of oxygen decline from an oxygen tension of 21% to 0.1% oxygen was explored using an incubator with programmable active control of gas concentrations. The effect of doping cultures with defined concentrations of RBCs was also used to evaluate the potential for RBCs to serve as a natural buffer in the setting of declining oxygen levels. RESULTS: A delay in onset of hypoxia over 96 hours resulted in a 3-fold increase in the relative colony forming efficiency (rCFE) of CTPs as compared to an immediate onset of hypoxia. The presence of RBCs in vitro inhibited the rCFE of CTPs. Given the negative effects of RBCs, methods of RBC removal were evaluated and compared for their effectiveness of RBC removal and retention of colony forming efficiency. CONCLUSIONS: These data suggest that conditions of hypoxia compromise colony forming efficiency in marrow derived CTPs. However, slowing the rate of decline of oxygen preserved colony forming efficiency at levels achieved in a stable normoxic (3% O2) environment. These data also suggest that RBCs are detrimental to the rCFE of CTPs and that buffy coat is an effective and preferred method for removing RBCs from marrow aspirates while preserving CTPs. These findings may inform clinical strategies for CTP transplantation.

Post microtextures accelerate cell proliferation and osteogenesis.

The influence of surface microtexture on osteogenesis was investigated in vitro by examining the proliferation and differentiation characteristics of a class of adult stem cells and their progeny, collectively known as connective tissue progenitor cells (CTPs). Human bone marrow-derived CTPs were cultured for up to 60 days on smooth polydimethylsiloxane (PDMS) surfaces and on PDMS with post microtextures that were 10 microm in diameter and 6 microm in height, with 10 microm separation. DNA quantification revealed that the numbers of CTPs initially attached to both substrates were similar. However, cells on microtextured PDMS transitioned from lag phase after 4 days of culture, in contrast to 6 days for cells on smooth surfaces. By day 9 cells on the smooth surfaces exhibited arbitrary flattened shapes and migrated without any preferred orientation. In contrast, cells on the microtextured PDMS grew along the array of posts in an orthogonal manner. By days 30 and 60 cells grew and covered all surfaces with extracellular matrix. Western blot analysis revealed that the expression of integrin alpha5 was greater on the microtextured PDMS compared with smooth surfaces. Real time reverse transcription-polymerase chain reaction revealed that gene expression of alkaline phosphatase had decreased by days 30 and 60, compared with that on day 9, for both substrates. Gene expression of collagen I and osteocalcin was consistently greater on post microtextures relative to smooth surfaces at all time points.

Transpedicular aspiration of osteoprogenitor cells from the vertebral body: progenitor cell concentrations affected by serial aspiration.

BACKGROUND CONTEXT: Spinal fusion is facilitated when the fusion site is augmented with autograft bone. Iliac crest, long the preferred source of autograft material, is the site of frequent complications and pain. Connective tissue progenitor cells (CTPs) aspirated from marrow provide a promising alternative to traditional autograft harvest. The vertebral body represents an even larger potential reservoir of progenitor cells than the ilium. PURPOSE: To test the hypothesis that a suitable concentration of osteoprogenitor cells can be aspirated from different depths of the vertebral body, maintaining progenitor cell concentrations comparable to the "gold standard," the iliac crest, even after sequential aspirations along the same transpedicular axis. STUDY DESIGN: Prospective clinical investigation quantifying CTP concentrations within the vertebral body relative to depth of sequential aspirations. PATIENT SAMPLE: Adult men and women undergoing elective posterior lumbar fusion and pedicle screw instrumentation (six men and seven women, mean age 56 years [range 40-74 years]). OUTCOME MEASURES: Cell count, CTP concentration (CTPs/cc marrow), and CTP prevalence (CTPs/million cells) were calculated for both individual and pooled aspirate samples. METHODS: Thirteen patients were enrolled into an institutional review board-approved protocol studying transpedicular aspiration of marrow progenitor cells. Connective tissue progenitor cells were aspirated from four depths along the transpedicular axis of the vertebral body and quantified according to cell concentration and CTP prevalence. Histochemical analysis of alkaline phosphatase-positive colony-forming units (CFUs) provided the prevalence of vertebral CTPs relative to depth of aspiration, vertebral level, age, and gender. RESULTS: Four 2.0cc aspirations were obtained from each pedicle of lumbar vertebrae selected for pedicle screw fixation (four 2.0cc aspirates from each of four pedicles). Aspirates of vertebral marrow demonstrated comparable or greater concentrations of CFUs compared with standards previously established for the iliac crest. Overall, the 208 aspirations from 26 vertebral bodies provided a mean CTP concentration of 741.5+/-976.2 CTPs per cubic centimeter of marrow, ranging from a mean concentration of 1316+/-1473 CTPs per cubic centimeter of marrow at superficial (30mm) aspirations to 439+/-557 CTPs per cubic centimeter marrow at deepest (45mm) aspiration depths (p<.00002). There were no significant differences relative to vertebral body level, side aspirated, or gender. An age-related decline in cellularity was suggested for vertebral body aspirates. CONCLUSIONS: The vertebral body is a potential marrow reservoir for aspiration of autograft osteogenic CTPs that can be used to augment spinal fusion. The cancellous bone within that portion of the vertebral body routinely cannulated during pedicle screw placement allows serial aspirations with only modest depletion of progenitor cell concentrations or dilution with peripheral blood. Connective tissue progenitor cell concentrations from all depths were comparable to the mean levels previously established for the iliac crest. The ability to simultaneously harvest progenitor cells for graft augmentation while preparing the pilot hole for pedicle screw fixation will expand the potential for cell harvest techniques for fusion augmentation and reduce the need for iliac crest harvest.

A three-dimensional scaffold with precise micro-architecture and surface micro-textures.

A three-dimensional (3D) structure comprising precisely defined micro-architecture and surface micro-textures, designed to present specific physical cues to cells and tissues, may provide an efficient scaffold in a variety of tissue engineering and regenerative medicine applications. We report a fabrication technique based on microfabrication and soft lithography that permits for the development of 3D scaffolds with both precisely engineered architecture and tailored surface topography. The scaffold fabrication technique consists of three key steps starting with microfabrication of a mold using an epoxy-based photoresist (SU-8), followed by dual-sided molding of a single layer of polydimethylsiloxane (PDMS) using a mechanical jig for precise motion control; and finally, alignment, stacking, and adhesion of multiple PDMS layers to achieve a 3D structure. This technique was used to produce 3D Texture and 3D Smooth PDMS scaffolds, where the surface topography comprised 10 microm diameter/height posts and smooth surfaces, respectively. The potential utility of the 3D microfabricated scaffolds, and the role of surface topography, were subsequently investigated in vitro with a combined heterogeneous population of adult human stem cells and their resultant progenitor cells, collectively termed connective tissue progenitors (CTPs), under conditions promoting the osteoblastic phenotype. Examination of bone-marrow derived CTPs cultured on the 3D Texture scaffold for 9 days revealed cell growth in three dimensions and increased cell numbers compared to those on the 3D Smooth scaffold. Furthermore, expression of alkaline phosphatase mRNA was higher on the 3D Texture scaffold, while osteocalcin mRNA expression was comparable for both types of scaffolds.

The influence of tethered epidermal growth factor on connective tissue progenitor colony formation.

Strategies to combine aspirated marrow cells with scaffolds to treat connective tissue defects are gaining increasing clinical attention and use. In situations such as large defects where initial survival and proliferation of transplanted connective tissue progenitors (CTPs) are limiting, therapeutic outcomes might be improved by using the scaffold to deliver growth factors that promote the early stages of cell function in the graft. Signaling by the epidermal growth factor receptor (EGFR) plays a role in cell survival and has been implicated in bone development and homeostasis. Providing epidermal growth factor (EGF) in a scaffold-tethered format may sustain local delivery and shift EGFR signaling to pro-survival modes compared to soluble ligand. We therefore examined the effect of tethered EGF on osteogenic colony formation from human bone marrow aspirates in the context of three different adhesion environments using a total of 39 donors. We found that tethered EGF, but not soluble EGF, increased the numbers of colonies formed regardless of adhesion background, and that tethered EGF did not impair early stages of osteogenic differentiation.

Modulating human connective tissue progenitor cell behavior on cellulose acetate scaffolds by surface microtextures.

Soft lithography techniques are used to fabricate cellulose acetate (CA) scaffolds with surface microtextures to observe growth characteristics of the progeny of human marrow-derived connective tissue progenitor cells (CTPs). Human CTPs were collected and cultured on CA scaffolds comprised postmicrotextures and smooth surfaces for up to 30 days. Cells on the smooth surfaces migrated without any preferred orientation for up to 30 days. On microtextures, cells tended to direct their processes toward posts and other cells on day 9. By day 30, cells on microtextures covered the surface with extracellular matrix. DNA quantification revealed approximately threefold more cells on microtextures than on the smooth surfaces. The alkaline phosphatase (AP) mRNA expression was slightly higher on smooth surfaces on day 9. However, by day 30, AP mRNA showed higher expression on microtextures. The mRNA expression of collagen type I was increased on microtextures by day 30, whereas smooth surfaces demonstrated similar expression. The osteocalcin mRNA expression was increased on postmicrotextures relative to smooth surfaces by day 30.

The effect of oxygen tension on the in vitro assay of human osteoblastic connective tissue progenitor cells.

Connective tissue progenitors (CTPs) are defined as the heterogeneous set of stem and progenitor cells that reside in native tissues and are capable of proliferation and differentiation into one or more connective tissue phenotypes. CTPs play important roles in tissue formation, repair, and remodeling. Therefore, in vitro assays of CTP prevalence and biological potential have important scientific and clinical relevance. This study evaluated oxygen tension as an important variable in optimizing in vitro conditions for quantitative assays of human CTPs. Bone marrow aspirates were collected from 20 human subjects and cultured using established medium conditions at ambient oxygen tensions of 1, 5, 10, and 20%. Colony-forming efficiency (CFE), proliferation, and colony density were assessed. CFE and proliferation were greatest at 5% O(2). Traditional conditions using atmospheric oxygen tension (20% O(2)) reduced CFE by as much as 32%. CFE and proliferation at 1% O(2) were less than 5% O(2) but comparable to that seen at 20% O(2), suggesting that CTPs are relatively resilient under hypoxic conditions, a fact that may be relevant to their function in wound repair and their potential use in tissue engineering applications involving transplantation into settings of moderate to severe hypoxia. These data demonstrate that optimization of quantitative assays for CTPs will require control of oxygen tension.

Formation of osteogenic colonies on well-defined adhesion peptides by freshly isolated human marrow cells.

Bone graft performance can be enhanced by addition of connective tissue progenitors (CTPs) from fresh bone marrow in a manner that concentrates the CTP cell population within the graft. Here, we used small peptide adhesion ligands presented against an otherwise adhesion-resistant synthetic polymer background in order to illuminate the molecular basis for the attachment and colony formation by osteogenic CTPs from fresh human marrow, and contrast the behavior of fresh marrow to many commonly used osteogenic cell sources. The linear GRGDSPY ligand was as effective as tissue culture polystyrene in fostering attachment of culture-expanded porcine CTPs. Although this GRGDSPY peptide was more effective than control peptides in fostering alkaline phosphatase (AP)-positive colony formation from primary human marrow in 5 of the 7 patients tested, GRGDSPY was as effective as the control glass substrate in only one patient of 7. Thus, the peptide appears capable of enabling osteoblastic development from only a subpopulation of CTPs in marrow. The bone sialoprotein-derived peptide FHRRIKA was ineffective in fostering attachment of primary culture-expanded pig CTPs, although it was as effective as GRGDSPY in fostering AP-positive colonies from fresh human marrow. This study provides insights into integrin-mediated behaviors of CTPs and highlights differences between freshly isolated marrow and culture-expanded cells.

Aspiration of osteoprogenitor cells for augmenting spinal fusion: comparison of progenitor cell concentrations from the vertebral body and iliac crest.

BACKGROUND: Successful arthrodesis in challenging clinical scenarios is facilitated when the site is augmented with autograft bone. The iliac crest has long been the preferred source of autograft material, but graft harvest is associated with frequent complications and pain. Connective tissue progenitor cells aspirated from the iliac crest and concentrated with allograft matrix and demineralized bone matrix provide a promising alternative to traditional autograft harvest. The vertebral body, an even larger reservoir of myeloproliferative cells, should provide progenitor cell concentrations similar to those of the iliac crest. METHODS: Twenty-one adults (eleven men and ten women with a mean age of 59 +/- 14 years) undergoing posterior lumbar arthrodesis and pedicle screw instrumentation underwent transpedicular aspiration of connective tissue progenitor cells. Aspirates were obtained from two depths within the vertebral body and were quantified relative to matched, bilateral aspirates from the iliac crest that were obtained from the same patient at the same time. Histochemical analysis was used to determine the prevalence of vertebral progenitor cells relative to the depth of aspiration, the vertebral level, age, and gender, as compared with the iliac crest standard. The cell count, progenitor cell concentration (cells/cc marrow), and progenitor cell prevalence (cells/million cells) were calculated. RESULTS: Aspirates of vertebral marrow demonstrated comparable or greater concentrations of progenitor cells compared with matched controls from the iliac crest. Progenitor cell concentrations were consistently higher than matched controls from the iliac crest (p = 0.05). The concentration of osteogenic progenitor cells was, on the average, 71% higher in the vertebral aspirates than in the paired iliac crest samples (p = 0.05). With the numbers available, there were no significant differences relative to vertebral body level, the side aspirated, the depth of aspiration, or gender. An age-related decline in cellularity was suggested for the iliac crest aspirates. CONCLUSIONS: The vertebral body is a suitable site for aspiration of bone marrow for graft augmentation during spinal arthrodesis.

Selective retention of bone marrow-derived cells to enhance spinal fusion.

Connective tissue progenitors can be concentrated rapidly from fresh bone marrow aspirates using some porous matrices as a surface for cell attachment and selective retention, and for creating a cellular graft that is enriched with respect to the number of progenitor cells. We evaluated the potential value of this method using demineralized cortical bone powder as the matrix. Matrix alone, matrix plus marrow, and matrix enriched with marrow cells were compared in an established canine spinal fusion model. Fusions were compared based on union score, fusion mass, fusion volume, and by mechanical testing. Enriched matrix grafts delivered a mean of 2.3 times more cells and approximately 5.6 times more progenitors than matrix mixed with bone marrow. The union score with enriched matrix was superior to matrix alone and matrix plus marrow. Fusion volume and fusion area also were greater with the enriched matrix. These data suggest that the strategy of selective retention provides a rapid, simple, and effective method for concentration and delivery of marrow-derived cells and connective tissue progenitors that may improve the outcome of bone grafting procedures in various clinical settings.