Identification and specification of the mouse skeletal stem cell.

How are skeletal tissues derived from skeletal stem cells? Here, we map bone, cartilage, and stromal development from a population of highly pure, postnatal skeletal stem cells (mouse skeletal stem cells, mSSCs) to their downstream progenitors of bone, cartilage, and stromal tissue. We then investigated the transcriptome of the stem/progenitor cells for unique gene-expression patterns that would indicate potential regulators of mSSC lineage commitment. We demonstrate that mSSC niche factors can be potent inducers of osteogenesis, and several specific combinations of recombinant mSSC niche factors can activate mSSC genetic programs in situ, even in nonskeletal tissues, resulting in de novo formation of cartilage or bone and bone marrow stroma. Inducing mSSC formation with soluble factors and subsequently regulating the mSSC niche to specify its differentiation toward bone, cartilage, or stromal cells could represent a paradigm shift in the therapeutic regeneration of skeletal tissues.

Intermediate term outcomes after transvaginal uterine-preserving surgery in women with uterovaginal prolapse.

INTRODUCTION AND HYPOTHESIS: There is growing interest in and performance of uterine-preserving prolapse repairs. We hypothesized that there would be no difference in pelvic organ prolapse (POP) recurrence 2 years following transvaginal uterosacral ligament hysteropexy (USLH) and sacrospinous ligament hysteropexy (SSLH). METHODS: This is a retrospective cohort study with a cross-sectional survey of women who underwent transvaginal uterine-preserving POP surgery from May 2016 to December 2017. Patients were included if they underwent either USLH or SSLH. POP recurrence was defined as a composite of subjective symptoms and/or retreatment. A cross-sectional survey was used to assess pelvic floor symptoms and patient satisfaction. RESULTS: A total of 47 women met the criteria. Mean age was 52.8 ± 12.5 years, and all had a preoperative POP-Q stage of 2 (55.3%) or 3 (44.7%). Thirty (63.8%) underwent SSLH and 17 (36.2%) underwent USLH. There were no differences in patient characteristics or perioperative data. There was no difference in composite recurrence (26.7% [8] vs 23.5% [4]) and retreatment (6.7% [2] vs 0%) retrospectively between SSLH and USLH groups at 22.6 months. Survey response rate was 80.9% (38) with a response time of 30.7 (28.0-36.6) months. The majority of patients (84.2%) reported POP symptom improvement, and both groups reported great satisfaction (89.5%). In respondents, 13.2% (5) reported subjective recurrence and 5.3% (2) underwent retreatment, with no differences between hysteropexy types. There were no differences in other pelvic floor symptoms. CONCLUSIONS: Although 1 in 4 women experienced subjective POP recurrence after transvaginal uterine-preserving prolapse repair and <5% underwent retreatment at 2 years, our results must be interpreted with caution given our small sample size. No differences in outcomes were identified between hysteropexy types; however, additional studies should be performed to confirm these findings. Both hysteropexy approaches were associated with great patient satisfaction.

Pelvic organ prolapse recurrence in young women undergoing vaginal and abdominal colpopexy.

INTRODUCTION AND HYPOTHESIS: While pelvic organ prolapse (POP) recurrence is believed to increase over time, outcomes of young women who undergo POP surgery are unclear. We hypothesized POP recurrence incidence among women <49 years would be higher after vaginal versus abdominal colpopexy. METHODS: This is a retrospective cohort study with a cross-sectional survey of women 18-49 years who underwent primary POP surgery from January 2003 to September 2013. Patients were identified by CPT codes for vaginal and abdominal colpopexy. POP recurrence was defined as vaginal bulge symptoms, POP retreatment (pessary or surgery) or both. Validated questionnaires were used to assess pelvic floor symptoms, patient satisfaction and improvement. RESULTS: Three hundred thirty-three women met the criteria. Mean age was 43 (+ 5.3) years; 29.1% (97) had an abdominal colpopexy and 70.9% (236) had a vaginal colpopexy. The recurrence incidence overall was 32.0% (31) in the abdominal group and 24.2% (57) in the vaginal group (p = 0.15), with a 10.3% (10) retreatment incidence in the abdominal group and 5.9% (14) in the vaginal group (p = 0.16). Forty-five percent (149) responded to the survey at a median time of 7.9 (3.1-15.2) years since surgery. The overall recurrence incidence in respondents was 13.7% (7) in the abdominal group and 15.3% (15) in the vaginal group (p = 0.80), with a retreatment incidence of 5.9% (3) in the abdominal and 5.2% (5) in the vaginal group (p = 0.85). CONCLUSIONS: One in four young women experienced subjective POP recurrence and/or retreatment, but only 5-10% underwent retreatment. There does not appear to be a difference in the incidence of recurrence between vaginal and abdominal colpopexy.

DEL1 protects against chondrocyte apoptosis through integrin binding.

BACKGROUND: Osteoarthritis (OA) is a debilitating disease process, affecting mobility and overall health of millions. Current treatment is for symptomatic relief and discovery of approaches to halt or reverse damage is imperative. Deletion of developmental endothelial locus-1 (Del1) has been shown to increase severity of OA in knockout mice. We examined the intracellular pathways involved in the ability of DEL1 to protect chondrocytes from apoptosis and anoikis and hypothesized that it functioned via integrin signaling. MATERIALS AND METHODS: Primary human chondrocytes were treated with various inducers of apoptosis, including anoikis, in the presence of added DEL1 or bovine serum albumin as control. Various inhibitors of integrin binding were examined for their effect on DEL1 activity. Downstream signaling pathway components were detected by immunoblotting. RESULTS: The addition of DEL1 protected chondrocytes from multiple inducers of apoptosis as measured by cell survival, terminal deoxynucleotidyl transferase dUTP nick end labeling and caspase 3/7 assays (P < 0.05). The effect of DEL1 was blocked by RGD peptides and by antibodies directed to integrin αVß3, but not by controls or antibody to integrin α1 (P < 0.05). Treatment with DEL1 promoted ERK and AKT activation when cells were attached, but only AKT activation under conditions of anoikis. CONCLUSIONS: DEL1 protected chondrocytes from apoptosis in response to activators of either the intrinsic or extrinsic pathways, and to anoikis. This effect was mediated primarily through integrin αVß3. This represents a therapeutic target for therapies to prevent cartilage degeneration in OA.

Inhibition of IRE1 results in decreased scar formation.

Wound healing is characterized by the production of large amounts of protein necessary to replace lost cellular mass and extracellular matrix. The unfolded protein response (UPR) is an important adaptive cellular response to increased protein synthesis. One of the main components of the UPR is IRE1, an endoplasmic reticulum transmembrane protein with endonuclease activity that produces the activated form of the transcription factor XBP1. Using luciferase reporter mice for Xbp1 splicing, we showed that IRE1 was up-regulated during excisional wound healing at the time in wound healing consistent with that of the proliferative phase, when the majority of protein synthesis for cellular proliferation and matrix deposition occurs. Furthermore, using a small molecule inhibitor of IRE1 we demonstrated that inhibition of IRE1 led to decreased scar formation in treated mice. Results were recapitulated in a hypertrophic scar mouse model. These data help provide a cellular pathway to target in the treatment of hypertrophic scarring and keloid disorders.

Loss of keratinocyte focal adhesion kinase stimulates dermal proteolysis through upregulation of MMP9 in wound healing.

OBJECTIVE: To investigate how epithelial mechanotransduction pathways impact wound repair. BACKGROUND: Mechanical forces are increasingly recognized to influence tissue repair, but their role in chronic wound pathophysiology remains unknown. Studies have shown that chronic wounds exhibit high levels of matrix metalloproteinase 9 (MMP9), a key proteolytic enzyme that regulates wound remodeling. We hypothesized that epithelial mechanosensory pathways regulated by keratinocyte-specific focal adhesion kinase (FAK) control dermal remodeling via MMP9. METHODS: A standard wound model was applied to keratinocyte-specific FAK knockout (KO) and control mice. Rates of wound healing were measured and tissue was obtained for histologic and molecular analyses. Transcriptional and immunoblot assays were used to assess the activation of FAK, intracellular kinases, and MMP9 in vitro. A cell suspension model was designed to validate the importance of FAK mechanosensing, p38, and MMP9 secretion in human cells. Biomechanical testing was utilized to evaluate matrix tensile properties in FAK KO and control wounds. RESULTS: Wound healing in FAK KO mice was significantly delayed compared with controls (closure at 15 days compared with 20 days, P = 0.0003). FAK KO wounds demonstrated decreased dermal thickness and collagen density. FAK KO keratinocytes exhibited overactive p38 and MMP9 signaling in vitro, findings recapitulated in human keratinocytes via the deactivation of FAK in the cell suspension model. Functionally, FAK KO wounds were significantly weaker and more brittle than control wounds, results consistent with the histologic and molecular analyses. CONCLUSIONS: Keratinocyte FAK is highly responsive to mechanical cues and may play a critical role in matrix remodeling via regulation of p38 and MMP9. These findings suggest that aberrant epithelial mechanosensory pathways may contribute to pathologic dermal proteolysis and wound chronicity.

A phase 1 trial utilizing TMI with fludarabine-melphalan in patients with hematologic malignancies undergoing second allo-SCT.

Relapse after allogeneic stem cell transplantation (allo-SCT) remains the primary cause of treatment failure. A second SCT can result in long-term survival in a subset of patients, but the relapse rate remains high. We conducted a single-center, phase 1, modified 3 + 3 dose-escalation study of the feasibility of combining intensity-modulated total marrow irradiation (IM-TMI) with fludarabine and melphalan for conditioning. Between December 2015 and May 2020, 21 patients with relapsed hematologic disease undergoing second or greater allo-SCT were treated with IM-TMI doses of 6 Gy, 9 Gy, or 12 Gy. Dose-limiting toxicity was defined as a grade 3 or higher treatment-related adverse event; mucositis was the primary dose-limiting toxicity. The median times to neutrophil and platelet engraftment were 10 and 18 days, respectively. The 1-year cumulative incidence of graft-versus-host disease was 65% (95% confidence interval CI, 38-83). The nonrelapse mortality at 2 years was 17% (95% CI, 4-39). Cumulative incidence of relapse at 2 years was 35% (95% CI, 13-58). Two-year progression-free survival and overall survival were 48% and 50%. We conclude that combining IM-TMI with fludarabine-melphalan is feasible. We recommend 12 Gy of IM-TMI with fludarabine-melphalan for second SCT, although 9 Gy may be used for older or underweight patients.

Compliance With Pelvic Floor Physical Therapy in Patients Diagnosed With High-Tone Pelvic Floor Disorders.

OBJECTIVE: The primary objective of this study was to describe patient compliance with pelvic floor physical therapy (PFPT) for high-tone pelvic floor disorders (HTPFD) and to compare patients who are compliant with prescribed therapy to those who are not. The secondary objective is to describe second-line treatments offered for HTPFD for returning patients. METHODS: This is a retrospective cohort study of women with a HTPFD who were prescribed PFPT at a tertiary care referral center. Patients were excluded if they had a primary diagnosis of urinary incontinence, had undergone prior PFPT, or if PFPT was part of preoperative planning. Noncompliance with PFPT was defined as not being formally discharged from therapy by the treating therapist. RESULTS: Data on PFPT compliance were available for 662 patients (87.3%). A total of 128 patients (19.4%) were fully compliant. Noncompliant patients were more likely to smoke and to have mental health disease compared with compliant patients (18% vs 8.7%, P = 0.01, and 50.4% vs 37.5%, P = 0.009, respectively). A total of 285 patients (43.1%) returned to their prescribing provider. Noncompliant patients were less likely to return to their provider: 63.4% versus 29.7%, P = <0.0001. Of the patients who returned, 183 (64.2%) were offered second-line treatment. CONCLUSIONS: Only 1 in 5 patients referred to PFPT for management of a high-tone pelvic floor disorder is compliant with the recommended therapy. Patients who are noncompliant are less likely to return to their prescribing provider, and less than half of referred patients return. Sixty percent of patients who return are offered second-line treatment.

Del1 Knockout Mice Developed More Severe Osteoarthritis Associated with Increased Susceptibility of Chondrocytes to Apoptosis.

OBJECTIVE: We identified significant expression of the matricellular protein, DEL1, in hypertrophic and mature cartilage during development. We hypothesized that this tissue-specific expression indicated a biological role for DEL1 in cartilage biology. METHODS: Del1 KO and WT mice had cartilage thickness evaluated by histomorphometry. Additional mice underwent medial meniscectomy to induce osteoarthritis, and were assayed at 1 week for apoptosis by TUNEL staining and at 8 weeks for histology and OA scoring. In vitro proliferation and apoptosis assays were performed on primary chondrocytes. RESULTS: Deletion of the Del1 gene led to decreased amounts of cartilage in the ears and knee joints in mice with otherwise normal skeletal morphology. Destabilization of the knee led to more severe OA compared to controls. In vitro, DEL1 blocked apoptosis in chondrocytes. CONCLUSION: Osteoarthritis is among the most prevalent diseases worldwide and increasing in incidence as our population ages. Initiation begins with an injury resulting in the release of inflammatory mediators. Excessive production of inflammatory mediators results in apoptosis of chondrocytes. Because of the limited ability of chondrocytes to regenerate, articular cartilage deteriorates leading to the clinical symptoms including severe pain and decreased mobility. No treatments effectively block the progression of OA. We propose that direct modulation of chondrocyte apoptosis is a key variable in the etiology of OA, and therapies aimed at preventing this important step represent a new class of regenerative medicine targets.

A distinct regulatory region of the Bmp5 locus activates gene expression following adult bone fracture or soft tissue injury.

Bone morphogenetic proteins (BMPs) are key signaling molecules required for normal development of bones and other tissues. Previous studies have shown that null mutations in the mouse Bmp5 gene alter the size, shape and number of multiple bone and cartilage structures during development. Bmp5 mutations also delay healing of rib fractures in adult mutants, suggesting that the same signals used to pattern embryonic bone and cartilage are also reused during skeletal regeneration and repair. Despite intense interest in BMPs as agents for stimulating bone formation in clinical applications, little is known about the regulatory elements that control developmental or injury-induced BMP expression. To compare the DNA sequences that activate gene expression during embryonic bone formation and following acute injuries in adult animals, we assayed regions surrounding the Bmp5 gene for their ability to stimulate lacZ reporter gene expression in transgenic mice. Multiple genomic fragments, distributed across the Bmp5 locus, collectively coordinate expression in discrete anatomic domains during normal development, including in embryonic ribs. In contrast, a distinct regulatory region activated expression following rib fracture in adult animals. The same injury control region triggered gene expression in mesenchymal cells following tibia fracture, in migrating keratinocytes following dorsal skin wounding, and in regenerating epithelial cells following lung injury. The Bmp5 gene thus contains an "injury response" control region that is distinct from embryonic enhancers, and that is activated by multiple types of injury in adult animals.

Enhancing stem cell survival in vivo for tissue repair.

The ability to use progenitor cells for regenerative medicine remains an evolving but elusive clinical goal. A serious obstacle towards widespread use of stem cells for tissue regeneration is the challenges that face these cells when they are placed in vivo into a wound for therapy. These environments are hypoxic, acidic, and have an upregulation of inflammatory mediators creating a region that is hostile towards cellular survival. Within this environment, the majority of progenitor cells undergo apoptosis prior to participating in lineage differentiation and cellular integration. In order to maximize the clinical utility of stem cells, strategies must be employed to increase the cell's ability to survive in vivo through manipulation of both the stem cell and the surrounding environment. This review focuses on current advances and techniques being used to increase in vivo stem cell survival for the purpose of tissue regeneration.