Sonic hedgehog suppresses penile remodeling after cavernous nerve injury and sustains long-term normal penis morphology.

BACKGROUND: Cavernous nerve (CN) injury, which occurs in prostatectomy and diabetic cases, initiates penile remodeling, including smooth muscle apoptosis and increased collagen in the corpora cavernosa, which are underlying causes of erectile dysfunction. Sonic hedgehog (SHH) is a critical regulator of penile smooth muscle, and SHH treatment suppresses corpora cavernosa remodeling that occurs with CN injury. AIM: We examine if SHH treatment by peptide amphiphile (PA) in the first week after CN injury is sufficient to prevent long-term penis remodeling and if apoptosis inhibitors also suppress penile remodeling. METHODS: Bilateral CN crush was performed on adult Sprague-Dawley rats (P115-120) that underwent 1 of 3 treatments with novel extended-release nanofiber PA hydrogels for delivery: SHH protein (n = 10), mouse serum albumin protein (control, n = 7), or caspase 3 inhibitor (AC-DEVD-CHO, n = 10). Rats were sacrificed after 18 to 24 days. Additional rats underwent CN injury (n = 6) or CN injury and SHH PA treatment for 2 and 4 days (n = 8) and included sham controls (n = 3) and nonsurgery controls (n = 3). OUTCOMES: Trichrome stain, hydroxyproline assay, and Western analysis for α-actin (smooth muscle) and GAPDH were performed to examine smooth muscle retention and collagen abundance. RESULTS: Smooth muscle decreased with CN injury. Corpora cavernosa showed increased smooth muscle at 2, 4, and 24 days after CN injury with SHH PA treatment in comparison with mouse serum albumin treatment among CN-injured controls. Caspase 3-inhibited penis demonstrated little smooth muscle preservation. Collagen was decreased 23% with SHH PA treatment (P < .001) at 18 to 24 days after CN injury. Collagen was unchanged with caspase 3 inhibitor treatment (P > .99). CLINICAL TRANSLATION: It is important to know that treatments given at the time of CN injury have a sustained effect on preserving penile architecture and thus erectile function, making them valuable for clinical translation. STRENGTHS AND LIMITATIONS: SHH PA treatment preserves penile smooth muscle after CN injury. Time points past 24 days were not examined, although penile remodeling takes place acutely after CN injury. Measurement of erectile function was not examined. CONCLUSIONS: SHH treatment by PA in the first week after CN injury is sufficient to suppress penile remodeling and to preserve penile smooth muscle over time, which is critical to prevent development of erectile dysfunction. There is a difference in the corpora cavernosa smooth muscle from proximal to distal in the penis of the Sprague-Dawley rat model. It is critical when examining therapy efficacy to ensure that comparable regions of the penis are analyzed. STATEMENT OF SIGNIFICANCE: In this study, we examine if suppression of apoptosis in penile smooth muscle in the first week after cavernous nerve injury is sufficient to preserve smooth muscle long-term.

SHH regulates penile morphology and smooth muscle through a mechanism involving BMP4 and GREM1.

BACKGROUND: The cavernous nerve (CN) is frequently damaged in prostatectomy and diabetic patients with erectile dysfunction (ED), initiating changes in penile morphology including an acute and intense phase of apoptosis in penile smooth muscle and increased collagen, which alter penile architecture and make corpora cavernosa smooth muscle less able to relax in response to neurotransmitters, resulting in ED. AIM: Sonic hedgehog (SHH) is a critical regulator of penile smooth muscle, and SHH treatment suppresses penile remodeling after CN injury through an unknown mechanism; we examine if part of the mechanism of how SHH preserves smooth muscle after CN injury involves bone morphogenetic protein 4 (BMP4) and gremlin1 (GREM1). METHODS: Primary cultures of smooth muscle cells were established from prostatectomy, diabetic, hypertension and Peyronie's (control) (N = 18) patients. Cultures were characterized by ACTA2, CD31, P4HB, and nNOS immunohistochemical analysis. Patient smooth muscle cell growth was quantified in response to BMP4 and GREM1 treatment. Adult Sprague Dawley rats underwent 1 of 3 surgeries: (1) uninjured or CN-injured rats were treated with BMP4, GREM1, or mouse serum albumin (control) proteins via Affi-Gel beads (N = 16) or peptide amphiphile (PA) (N = 26) for 3 and 14 days, and trichrome stain was performed; (2) rats underwent sham (N = 3), CN injury (N = 9), or CN injury and SHH PA treatment for 1, 2, and 4 days (N = 9). OUTCOMES: Western analysis for BMP4 and GREM1 was performed; (3) rats were treated with 5E1 SHH inhibitor (N = 6) or IgG (control; N = 6) for 2 and 4 days, and BMP4 and GREM1 localization was examined. Statistics were performed by analysis of variance with Scheffé's post hoc test. RESULTS: BMP4 increased patient smooth muscle cell growth, and GREM1 decreased growth. In rats, BMP4 treatment via Affi-Gel beads and PA increased smooth muscle at 3 and 14 days of treatment. GREM1 treatment caused increased collagen and smooth muscle at 3 days, which switched to primarily collagen at 14 days. CN injury increased BMP4 and GREM1, while SHH PA altered Western band size, suggesting alternative cleavage and range of BMP4 and GREM1 signaling. SHH inhibition in rats increased BMP4 and GREM1 in fibroblasts. CLINICAL IMPLICATIONS: Understanding how SHH PA preserves and regenerates penile morphology after CN injury will aid development of ED therapies. STRENGTHS AND LIMITATIONS: SHH treatment alters BMP4 and GREM1 localization and range of signaling, which can affect penile morphology. CONCLUSION: Part of the mechanism of how SHH regulates corpora cavernosa smooth muscle involves BMP4 and GREM1.

BMP4 and GREM1 are targets of SHH signaling and downstream regulators of collagen in the penis.

BACKGROUND: Cavernous nerve (CN) injury, caused by prostatectomy and diabetes, initiates a remodeling process (smooth muscle apoptosis and increased collagen) in the corpora cavernosa of the penis of patients and animal models that is an underlying cause of erectile dysfunction (ED), and the Sonic hedgehog (SHH) pathway plays an essential role in the response of the penis to denervation, as collagen increases with SHH inhibition and decreases with SHH treatment. AIM: We examined if part of the mechanism of how SHH prevents penile remodeling and increased collagen with CN injury involves bone morphogenetic protein 4 (BMP4) and gremlin1 (GREM1) and examined the relationship between SHH, BMP4, GREM1, and collagen in penis of ED patients and rat models of CN injury, SHH inhibition, and SHH, BMP4, and GREM1 treatment. METHODS: Corpora cavernosa of Peyronie's disease (control), prostatectomy, and diabetic ED patients were obtained (N = 30). Adult Sprague Dawley rats (n = 90) underwent (1) CN crush (1-7 days) or sham surgery; (2) CN injury and BMP4, GREM1, or mouse serum albumin (control) treatment via Affi-Gel beads or peptide amphiphile (PA) for 14 days; (3) 5E1 SHH inhibitor, IgG, or phosphate-buffered saline (control) treatment for 2 to 4 days; or (4) CN crush with mouse serum albumin or SHH for 9 days. OUTCOMES: Immunohistochemical and Western analysis for BMP4 and GREM1, and collagen analysis by hydroxyproline and trichrome stain were performed. RESULTS: BMP4 and GREM1 proteins were identified in corpora cavernosa smooth muscle of prostatectomy, diabetic, and Peyronie's patients, and in rat smooth muscle, sympathetic nerve fibers, perineurium, blood vessels, and urethra. Collagen decreased 25.4% in rats with CN injury and BMP4 treatment (P = .02) and increased 61.3% with CN injury and GREM1 treatment (P = .005). Trichrome stain showed increased collagen in rats treated with GREM1. Western analysis identified increased BMP4 and GREM1 in corpora cavernosa of prostatectomy and diabetic patients, and after CN injury (1-2 days) in our rat model. Localization of BMP4 and GREM1 changed with SHH inhibition. SHH treatment increased the monomer form of BMP4 and GREM1, altering their range of signaling. CLINICAL IMPLICATIONS: A better understanding of penile remodeling and how fibrosis occurs with loss of innervation is essential for development of novel ED therapies. STRENGTHS AND LIMITATIONS: The relationship between SHH, BMP4, GREM1, and collagen is complex in the penis. CONCLUSION: BMP4 and GREM1 are downstream targets of SHH that impact collagen and may be useful in collaboration with SHH to prevent penile remodeling and ED.

Effect of Material and Pad Abrasion on Shear Bond Strength of 3D-Printed Orthodontic Brackets.

OBJECTIVE: To investigate the effect of printing material and air abrasion of bracket pads on the shear bond strength of 3D-printed plastic orthodontic brackets when bonded to the enamel of extracted human teeth. MATERIALS AND METHODS: Premolar brackets were 3D-printed using the design of a commercially available plastic bracket in two biocompatible resins: Dental LT Resin and Dental SG Resin (n = 40/material). 3D-printed brackets and commercially manufactured plastic brackets were divided into two groups (n = 20/group), one of which was air abraded. All brackets were bonded to extracted human premolars, and shear bond strength tests were performed. The failure types of each sample were classified using a 5-category modified adhesive remnant index (ARI) scoring system. RESULTS: Bracket material and bracket pad surface treatment presented statistically significant effects for shear bond strengths, and a significant interaction effect between bracket material and bracket pad surface treatment was observed. The non-air abraded (NAA) SG group (8.87 ± 0.64 MPa) had a statistically significantly lower shear bond strength than the air abraded (AA) SG group (12.09 ± 1.23 MPa). In the manufactured brackets and LT Resin groups, the NAA and AA groups were not statistically significantly different within each resin. A significant effect of bracket material and bracket pad surface treatment on ARI score was observed, but no significant interaction effect between bracket material and pad treatment was found. CONCLUSION: 3D-printed orthodontic brackets presented clinically sufficient shear bond strengths both with and without AA prior to bonding. The effect of bracket pad AA on shear bond strength depends on the bracket material.

Sonic Hedgehog Signaling in Primary Culture of Human Corpora Cavernosal Tissue From Prostatectomy, Diabetic, and Peyronie's Patients.

BACKGROUND: Cavernous nerve (CN) injury causes penile remodeling, including smooth muscle apoptosis and increased collagen, which results in erectile dysfunction (ED), and prevention of this remodeling is critical for novel ED therapy development. AIM: We developed 2 peptide amphiphile (PA) hydrogel delivery vehicles for Sonic hedgehog (SHH) protein to the penis and CN, which effectively suppress penile distrophic remodeling (apoptosis and fibrosis), in vivo in a rat CN injury model, and the aim of this study is to determine if SHH PA can be used to regenerate human corpora cavernosal smooth muscle deriving from multiple ED origins. METHODS: Corpora cavernosal tissue was obtained from prostatectomy, diabetic, hypertension, cardiovascular disease and Peyronie's (control) patients (n = 21). Primary cultures (n = 21) were established, and corpora cavernosal cells were treated with SHH protein, MSA (control), 5E1 SHH inhibitor, and PBS (control). Growth was quantified by counting the number of cells at 3-4 days. Statistics were performed by ANOVA with Scheffe's post hoc test. Concentration of SHH protein for maximal growth was optimized, and a more active SHH protein examined. OUTCOMES: Cultures were characterized by immunohistochemical analysis with ACTA2, CD31, nNOS and P4HB, and smooth muscle was quantified in comparison to DAPI. RESULTS: Cultures established were >97% smooth muscle. SHH protein increased growth of smooth muscle cells from prostatectomy, diabetic, and Peyronie's patients in a similar manner (49%-51%), and SHH inhibition decreased growth (20%-33%). There was no difference in growth using 25 ug and 10 ug SHH protein, suggesting a threshold concentration of SHH protein above which smooth muscle growth is enhanced. A more active lipid modified SHH peptide further enhanced growth (15%), indicating a more robust growth response. SHH increased growth in smooth muscle cells from hypertension (37%) and cardiovascular disease (32%) patients. SHH protein increased growth under normal and high glucose conditions, suggesting that high glucose conditions that may be present in under controlled diabetic patients would not detract from SHH regenerative capacity. CLINICAL IMPLICATIONS: SHH PA would be beneficial to enhance smooth muscle regeneration in patients with ED of multiple etiologies. STRENGTHS AND LIMITATIONS: Understanding how human corpora cavernosal tissue responds to SHH treatment is critical for clinical translation of SHH PA to ED patients. CONCLUSION: Corpora cavernosal smooth muscle from all ED patients responded to SHH treatment with increased growth. Stupp, SI. Sonic Hedgehog Signaling in Primary Culture of Human Corpora Cavernosal Tissue From Prostatectomy, Diabetic, and Peyronie's Patients. J Sex Med 2022;19:1228-1242.

Caspase Signaling in ED Patients and Animal Models.

BACKGROUND: Current treatments for erectile dysfunction (ED) are ineffective in prostatectomy and diabetic patients due to cavernous nerve (CN) injury, which causes smooth muscle apoptosis, penile remodeling, and ED. Apoptosis can occur via the intrinsic (caspase 9) or extrinsic (caspase 8) pathway. AIM: We examined the mechanism of how apoptosis occurs in ED patients and CN injury rat models to determine points of intervention for therapy development. METHODS AND OUTCOMES: Immunohistochemical and western analyses for caspase 3-cleaved, caspase-8 and caspase-9 (pro and active forms) were performed in corpora cavernosal tissue from Peyronie's, prostatectomy and diabetic ED patients (n = 33), penis from adult Sprague Dawley rats that underwent CN crush (n = 24), BB/WOR diabetic and control rats (n = 8), and aged rats (n = 9). RESULTS: Caspase 3-cleaved was observed in corpora cavernosa from Peyronie's patients and at higher abundance in prostatectomy and diabetic tissues. Apoptosis takes place primarily through the extrinsic (caspase 8) pathway in penis tissue of ED patients. In the CN crushed rat, caspase 3-cleaved was abundant from 1-9 days after injury, and apoptosis takes place primarily via the intrinsic (caspase 9) pathway. Caspase 9 was first observed and most abundant in a layer under the tunica, and after several days was observed in the lining of and between the sinuses of the corpora cavernosa. Caspase 8 was initially observed at low abundance in the rat corpora cavernosa and was not observed at later time points after CN injury. Aged and diabetic rat penis primarily exhibited intrinsic mechanisms, with diabetic rats also exhibiting mild extrinsic activation. CLINICAL TRANSLATION: Knowing how and when to intervene to prevent the apoptotic response most effectively is critical for the development of drugs to prevent ED, morphological remodeling of the corpora cavernosa, and thus, disease management. STRENGTHS AND LIMITATIONS: Animal models may diverge from the signaling mechanisms observed in ED patients. While the rat utilizes primarily caspase 9, there is a significant flux through caspase 8 early on, making it a reasonable model, as long as the timing of apoptosis is considered after CN injury. CONCLUSIONS: Apoptosis takes place primarily through the extrinsic caspase 8 dependent pathway in ED patients and via the intrinsic caspase 9 dependent pathway in commonly used CN crush ED models. This is an important consideration for study design and interpretation that must be taken into account for therapy development and testing of drugs, and our therapeutic targets should ideally inhibit both apoptotic mechanisms. Martin S, Harrington DA, Ohlander S, et al. Caspase Signaling in ED Patients and Animal Models. J Sex Med 2021;18:711-722.

Functional characterization of ATF1, GREM2 AND WNT10B variants associated with tooth agenesis.

OBJECTIVE: To determine the functional effects of ATF1, WNT10B and GREM2 gene variants identified in individuals with tooth agenesis (TA). SETTINGS AND SAMPLE POPULATION: Stem cells from human exfoliated deciduous teeth (SHED) were used as an in vitro model system to test the effect of TA-associated variants. MATERIALS AND METHODS: Plasmid constructs containing reference and mutant alleles for ATF1 rs11169552, WNT10B rs833843 and GREM2 rs1414655 variants were transfected into SHED for functional characterization of variants. Allele-specific changes in gene transcription activity, protein expression, cell migration and proliferation, and expression of additional tooth development genes (MSX1, PAX9 and AXIN2) were evaluated. Data analyses were performed using Student's t-test. P-values ≤ .05 were considered statistically significant. RESULTS: Mutant variants resulted in significantly decreased transcriptional activity of respective genes (P < 0.05), although no changes in protein localization were noted. Expression of MSX1 was significantly decreased in ATF1- and GREM2-mutant cells, whereas PAX9 or AXIN2 mRNA expression was not significantly altered. Mutant WNT10B had no significant effect on the expression of additional TA genes. ATF1- and GREM2-mutant cells presented increased cell migration. Cell proliferation was also affected with all three mutant alleles. CONCLUSIONS: Our results demonstrate that ATF1, WNT10B and GREM2 mutant alleles have modulatory effects on gene/protein function that may contribute to TA.

Peptide amphiphile nanofiber hydrogel delivery of Sonic hedgehog protein to the penis and cavernous nerve suppresses intrinsic and extrinsic apoptotic signaling mechanisms, which are an underlying cause of erectile dysfunction.

Erectile dysfunction (ED) is a common and debilitating condition with high impact on quality of life. An underlying cause of ED is apoptosis of penile smooth muscle, which occurs with cavernous nerve injury, in prostatectomy, diabetic and aging patients. We are developing peptide amphiphile (PA) nanofiber hydrogels as an in vivo delivery vehicle for Sonic hedgehog protein to the penis and cavernous nerve to prevent the apoptotic response. We examine two important aspects required for clinical application of the biomaterials: if SHH PA suppresses intrinsic (caspase 9) and extrinsic (caspase 8) apoptotic mechanisms, and if suppressing one apoptotic mechanism forces apoptosis to occur via a different mechanism. We show that SHH PA suppresses both caspase 9 and 8 apoptotic mechanisms, and suppressing caspase 9 did not shift signaling to caspase 8. SHH PA has significant clinical potential as a preventative ED therapy, by management of intrinsic and extrinsic apoptotic mechanisms.

Effect of surface preparation and light curing on penetration of silver particles from 38% silver diamine fluoride in dentin of primary teeth: An in vitro evaluation.

PURPOSE: To evaluate the effect of light cure, as well as various dentin surface treatment approaches, on the penetration depth of silver precipitating from 38% silver diamine fluoride into primary dentin tubules. METHODS: The occlusal dentin surfaces of 42 non-carious primary molars were exposed and then sectioned into halves bucco-lingually. The halves from each tooth pair were randomly split in two mega-groups, and each mega-group was divided randomly as follows into six experimental groups: prepared by either carbide bur (G1, G2), ceramic bur (G3, G4), or erbium laser (G5, G6). SDF was then applied to all prepared surfaces, and finally even-numbered groups (G2, G4, G6) were light cured. One mega-group was assigned to quantitative evaluation of silver penetration depth along the axial wall, and the other mega-group was reserved for qualitative observation of relative silver distribution on the occlusal surface, both via scanning electron microscope. RESULTS: No significant difference was observed in silver penetration depth between light cure and non-light cure groups (P= 0.8908). There was a statistically significant association between tooth preparation method and depth of silver penetration (P< 0.000001); laser-treated groups had significantly deeper silver penetration (1,148.9 µm G5, 1160.4 µm G6) than carbide bur (P< 0.05; 184.7 µm G1, 301.8 µm G2) or ceramic bur (P< 0.05; 184.1 µm G3, 131.0 µm G4) groups. A significant difference (P< 0.05) was noted in percentage occlusal surface coverage of particles between laser (51.4% G5, 35.8% G6) and carbide groups (21.1% G1, 19.3% G2). Light cure had no significant effect on the depth of silver penetration from 38% SDF in the dentin of primary teeth. Laser preparation resulted in deeper silver penetration than carbide or ceramic bur. CLINICAL SIGNIFICANCE: Exposure of 38% silver diamine fluoride-treated dentin to light cure did not affect the depth of penetration of silver particles into the dentin tubules of primary teeth. Rather, tooth preparation approaches that reduce the smear layer, like laser ablation, resulted in the deepest penetration of silver into the tubules. Clinical application of these findings will depend on scenario and treatment aim.

Optimization of Sonic Hedgehog Delivery to the Penis from Self-Assembling Nanofiber Hydrogels to Preserve Penile Morphology after Cavernous Nerve Injury.

Erectile dysfunction (ED) is a significant medical condition, with high impact on patient quality of life. Current treatments are minimally effective in prostatectomy, diabetic and aging patients due to injury to the cavernous nerve (CN); loss of innervation causes extensive smooth muscle (SM) apoptosis, increased collagen and ED. Sonic hedgehog (SHH) is a critical regulator of penile SM. We developed a self-assembling peptide amphiphile (PA) nanofiber hydrogel for extended release of SHH protein to the penis after CN injury, to suppress SM apoptosis. In this study we optimize the animal model, SHH concentration, duration of suppression, and location of delivery, to maximize SM preservation. SHH treatment suppressed apoptosis and preserved SM 48%. Increased SHH duration preserved SM 100%. Simultaneous penis/CN delivery increased SM 127%. Optimization of SHH PA delivery is essential for clinical translation to ED patients, and the PA vehicle has wide applicability as an in vivo delivery tool.

Sonic hedgehog regulation of human rhabdosphincter muscle:Potential implications for treatment of stress urinary incontinence.

AIMS: Rhabdosphincter (RS) muscle injury occurs during prostatectomy, and is a leading cause of stress urinary incontinence (SUI). Current SUI treatments engender significant side effects, which negatively impact patient quality of life. Thus an unmet need exists to develop novel RS regeneration methods. We have shown that Sonic hedgehog (SHH) is a critical regulator of penile smooth muscle, and we have developed novel peptide amphiphile nanofiber hydrogel delivery of SHH protein to the penis to regenerate smooth muscle after prostatectomy induced injury. If similar SHH signaling mechanisms regulate RS muscle homeostasis, this innovative technology may be adapted for RS regeneration post-prostatectomy. We examine the SHH pathway in human RS muscle. METHODS: Human RS obtained during radical cystoprostatectomy (n = 13), underwent SHH pathway analysis. Primary cultures were established (n = 5), and RS cells were treated with SHH protein, SHH inhibitor, or PBS (control). Immunohistochemical analysis for SHH pathway, skeletal muscle actin, and trichrome stain were performed. RS growth was quantified at 3 and 6 days. RESULTS: SHH, it is receptors patched and smoothened, and transcriptional activators, GLI proteins, were identified in human RS muscle. At 3 and 6 days, RS cells increased 62% and 78% (P = 0.0001) with SHH treatment and decreased 40% (P = 0.0001) and 18% (P = 0.039) with SHH inhibition. CONCLUSIONS: The SHH pathway was identified in human RS. RS growth increased with SHH treatment, indicating intervention may be possible to enhance RS regeneration, and impact SUI. Peptide amphiphile delivery of SHH may be applicable for RS regeneration and SUI prevention.

Peptide amphiphile delivery of sonic hedgehog protein promotes neurite formation in penile projecting neurons.

Erectile dysfunction (ED) critically impacts quality of life in prostatectomy, diabetic and aging patients. The underlying mechanism involves cavernous nerve (CN) damage, resulting in ED in 80% of prostatectomy patients. Peptide amphiphile (PA) nanofiber hydrogel delivery of sonic hedgehog (SHH) protein to the injured CN, improves erectile function by 60% at 6 weeks after injury, by an unknown mechanism. We hypothesize that SHH is a regulator of neurite formation. SHH treatment promoted extensive neurite formation in uninjured and crushed CNs, and SHH inhibition decreased neurites >80%. Most abundant neurites were observed with continuous SHH PA treatment of crushed CNs. Once induced with SHH, neurites continued to grow. SHH rescued neurite formation when not given immediately. SHH is a critical regulator of neurite formation in peripheral neurons under uninjured and regenerative conditions, and SHH PA treatment at the time of injury/prostatectomy provides an exploitable avenue for intervention to prevent ED.

Peptide amphiphile nanofiber hydrogel delivery of sonic hedgehog protein to the cavernous nerve to promote regeneration and prevent erectile dysfunction.

Erectile dysfunction (ED) has high impact on quality of life in prostatectomy, diabetic and aging patients. An underlying mechanism is cavernous nerve (CN) injury, which causes ED in up to 80% of prostatectomy patients. We examine how sonic hedgehog (SHH) treatment with innovative peptide amphiphile nanofiber hydrogels (PA), promotes CN regeneration after injury. SHH and its receptors patched (PTCH1) and smoothened (SMO) are localized in PG neurons and glia. SMO undergoes anterograde transport to signal to downstream targets. With crush injury, PG neurons degenerate and undergo apoptosis. SHH protein decreases, SMO localization changes to the neuronal cell surface, and anterograde transport stops. With SHH treatment SHH is taken up at the injury site and undergoes retrograde transport to PG neurons, allowing SMO transport to occur, and neurons remain intact. SHH treatment prevents neuronal degeneration, maintains neuronal, glial and downstream target signaling, and is significant as a regenerative therapy.

Hydrogel-based 3D model of patient-derived prostate xenograft tumors suitable for drug screening.

The lack of effective therapies for bone metastatic prostate cancer (PCa) underscores the need for accurate models of the disease to enable the discovery of new therapeutic targets and to test drug sensitivities of individual tumors. To this end, the patient-derived xenograft (PDX) PCa model using immunocompromised mice was established to model the disease with greater fidelity than is possible with currently employed cell lines grown on tissue culture plastic. However, poorly adherent PDX tumor cells exhibit low viability in standard culture, making it difficult to manipulate these cells for subsequent controlled mechanistic studies. To overcome this challenge, we encapsulated PDX tumor cells within a three-dimensional hyaluronan-based hydrogel and demonstrated that the hydrogel maintains PDX cell viability with continued native androgen receptor expression. Furthermore, a differential sensitivity to docetaxel, a chemotherapeutic drug, was observed as compared to a traditional PCa cell line. These findings underscore the potential impact of this novel 3D PDX PCa model as a diagnostic platform for rapid drug evaluation and ultimately push personalized medicine toward clinical reality.

Microfluidic coaxial 3D bioprinting of cell-laden microfibers and microtubes for salivary gland tissue engineering.

Replacement therapy for the salivary gland (SG) remains an unmet clinical need. Xerostomia ("dry mouth") due to hyposalivation can result from injury or disease to the SG, such as salivary acinar death caused by radiation therapy (RT) for head and neck squamous cell carcinoma (HNSCC). Currently, only palliative treatments exist for xerostomia, and many patients endure deteriorated oral health and poor quality of life. Tissue engineering could offer a permanent solution for SG replacement by isolating healthy SG tissues prior to RT, expanding its cells in vitro, and recreating a functional salivary neogland for implantation post-RT. 3D bioprinting methods potentiate spatial cell deposition into defined hydrogel-based architectures, mimicking the thin epithelia developed during the complex branching morphogenesis of SG. By leveraging a microfluidics-based bioprinter with coaxial polymer and crosslinker streams, we fabricated thin, biocompatible, and reproducible hydrogel features that recapitulate the thin epithelia characteristics of SG. This flexible platform enabled two modes of printing: we produced solid hydrogel fibers, with diameters <100 µm, that could be rastered to create larger mm-scale structures. By a second method, we generated hollow tubes with wall thicknesses ranging 45-80 µm, total tube diameters spanning 0.6-2.2 mm, and confirmed tube patency. In both cases, SG cells could be printed within the thin hydrogel features, with preserved phenotype and high viability, even at high density (5.0 × 106 cells/mL). Our work demonstrates hydrogel feature control across multiple length scales, and a new paradigm for addressing SG restoration by creating microscale tissue engineered components.

A 3D Perfusable Platform for In Vitro Culture of Patient Derived Xenografts.

Many advanced cancer models, such as patient-derived xenografts (PDXs), offer significant benefits in their preservation of the native tumor's heterogeneity and susceptibility to treatments, but face significant barriers to use in their reliance on a rodent host for propagation and screening. PDXs remain difficult to implement in vitro, particularly in configurations that enable both detailed cellular analysis and high-throughput screening (HTS). Complex multilineage co-cultures with stromal fibroblasts, endothelium, and other cellular and structural components of the tumor microenvironment (TME) further complicate ex vivo implementation. Herein, the culture of multiple prostate cancer (PCa)-derived PDX models as 3D clusters within engineered biomimetic hydrogel matrices, in a HTS-compatible multiwell microfluidic format, alongside bone marrow-derived stromal cells and a perfused endothelial channel. Polymeric hydrogel matrices are customized for each cell type, enabling cell survival in vitro and facile imaging across all conditions. PCa PDXs demonstrate unique morphologies and reliance on TME partners, retention of known phenotype, and expected sensitivity or resistance to standard PCa therapeutics. This novel integration of technologies provides a fully human model, and expands the information to be gathered from each specimen, while avoiding the time and labor involved with animal-based testing.

Characterization and functionality of cardiac progenitor cells in congenital heart patients.

BACKGROUND: Human cardiac progenitor cells (hCPCs) may promote myocardial regeneration in adult ischemic myocardium. The regenerative capacity of hCPCs in young patients with nonischemic congenital heart defects for potential use in congenital heart defect repair warrants exploration. METHODS AND RESULTS: Human right atrial specimens were obtained during routine congenital cardiac surgery across 3 groups: neonates (age, <30 days), infants (age, 1 month to 2 years), and children (age, >2 to ≤13 years). C-kit(+) hCPCs were 3-fold higher in neonates than in children >2 years of age. hCPC proliferation was greatest during the neonatal period as evidenced by c-kit(+) Ki67(+) expression but decreased with age. hCPC differentiation capacity was also greatest in neonatal right atrium as evidenced by c-kit(+), NKX2-5(+), NOTCH1(+), and NUMB(+) expression. Despite the age-dependent decline in resident hCPCs, we isolated and expanded right atrium-derived CPCs from all patients (n=103) across all ages and diagnoses using the cardiosphere method. Intact cardiospheres contained a mix of heart-derived cell subpopulations that included cardiac progenitor cells expressing c-kit(+), Islet-1, and supporting cells. The number of c-kit(+)-expressing cells was highest in human cardiosphere-derived cells (hCDCs) grown from neonatal and infant right atrium. Furthermore, hCDCs could differentiate into diverse cardiovascular lineages by in vitro differentiation assays. Transplanted hCDCs promoted greater myocardial regeneration and functional improvement in infarcted myocardium than transplanted cardiac fibroblasts. CONCLUSIONS: Resident hCPCs are most abundant in the neonatal period and rapidly decrease over time. hCDCs can be reproducibly isolated and expanded from young human myocardial samples regardless of age or diagnosis. hCPCs are functional and have potential in congenital cardiac repair.

Hyaluronic Acid-Based Hydrogels: from a Natural Polysaccharide to Complex Networks.

Hyaluronic acid (HA) is one of nature's most versatile and fascinating macromolecules. Being an essential component of the natural extracellular matrix (ECM), HA plays an important role in a variety of biological processes. Inherently biocompatible, biodegradable and non-immunogenic, HA is an attractive starting material for the construction of hydrogels with desired morphology, stiffness and bioactivity. While the interconnected network extends to the macroscopic level in HA bulk gels, HA hydrogel particles (HGPs, microgels or nanogels) confine the network to microscopic dimensions. Taking advantage of various scaffold fabrication techniques, HA hydrogels with complex architecture, unique anisotropy, tunable viscoelasticity and desired biologic outcomes have been synthesized and characterized. Physical entrapment and covalent integration of hydrogel particles in a secondary HA network give rise to hybrid networks that are hierarchically structured and mechanically robust, capable of mediating cellular activities through the spatial and temporal presentation of biological cues. This review highlights recent efforts in converting a naturally occurring polysaccharide to drug releasing hydrogel particles, and finally, complex and instructive macroscopic networks. HA-based hydrogels are promising materials for tissue repair and regeneration.

Bringing hydrogel-based craniofacial therapies to the clinic.

This review explores the evolution of the use of hydrogels for craniofacial soft tissue engineering, ranging in complexity from acellular injectable fillers to fabricated, cell-laden constructs with complex compositions and architectures. Addressing both in situ and ex vivo approaches, tissue restoration secondary to trauma or tumor resection is discussed. Beginning with relatively simple epithelia of oral mucosa and gingiva, then moving to more functional units like vocal cords or soft tissues with multilayer branched structures, such as salivary glands, various approaches are presented toward the design of function-driven architectures, inspired by native tissue organization. Multiple tissue replacement paradigms are presented here, including the application of hydrogels as structural materials and as delivery platforms for cells and/or therapeutics. A practical hierarchy is proposed for hydrogel systems in craniofacial applications, based on their material and cellular complexity, spatial order, and biological cargo(s). This hierarchy reflects the regulatory complexity dictated by the Food and Drug Administration (FDA) in the United States prior to commercialization of these systems for use in humans. The wide array of available biofabrication methods, ranging from simple syringe extrusion of a biomaterial to light-based spatial patterning for complex architectures, is considered within the history of FDA-approved commercial therapies. Lastly, the review assesses the impact of these regulatory pathways on the translational potential of promising pre-clinical technologies for craniofacial applications. STATEMENT OF SIGNIFICANCE: While many commercially available hydrogel-based products are in use for the craniofacial region, most are simple formulations that either are applied topically or injected into tissue for aesthetic purposes. The academic literature previews many exciting applications that harness the versatility of hydrogels for craniofacial soft tissue engineering. One of the most exciting developments in the field is the emergence of advanced biofabrication methods to design complex hydrogel systems that can promote the functional or structural repair of tissues. To date, no clinically available hydrogel-based therapy takes full advantage of current pre-clinical advances. This review surveys the increasing complexity of the current landscape of available clinical therapies and presents a framework for future expanded use of hydrogels with an eye toward translatability and U.S. regulatory approval for craniofacial applications.

Peptide amphiphile nanofiber delivery of sonic hedgehog protein to reduce smooth muscle apoptosis in the penis after cavernous nerve resection.

INTRODUCTION: Erectile dysfunction (ED) is a serious medical condition that affects 16-82% of prostate cancer patients treated by radical prostatectomy and current treatments are ineffective in 50-60% of prostatectomy patients. The reduced efficacy of treatments makes novel therapeutic approaches to treat ED essential. The secreted protein Sonic hedgehog (SHH) is a critical regulator of penile smooth muscle and apoptosis that is decreased in cavernous nerve (CN) injury and diabetic ED models. Past studies using Affi-Gel beads have shown SHH protein to be effective in suppressing apoptosis caused by CN injury. AIM: We hypothesize that SHH protein delivered via novel peptide amphiphile (PA) nanofibers will be effective in suppressing CN injury-induced apoptosis. METHODS: Adult Sprague Dawley rats (n=50) were used to optimize PA injection in vivo. PA with SHH protein (n=16) or bovine serum albumin (BSA) (control, n=14) was injected into adult rats that underwent bilateral CN cut. Rats were sacrificed at 2, 4, and 7 days. Alexa Fluor-labeled SHH protein was used to determine the target of SHH signaling (n=3). MAIN OUTCOME MEASURES: Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and semiquantitative immunohistochemical analysis for SHH protein and cluster differentiation protein three (CD3) were performed. RESULTS: SHH-PA caused a 25% and 16% reduction in apoptosis at 4 and 7 days after CN injury and a 9.3% and 19% increase in SHH protein at 4 and 7 days after CN injury. CD3 protein was not observed in SHH-PA-treated penis. In vitro, 73% of SHH protein diffused from PA within 6 days. Labeled SHH was observed in smooth muscle. CONCLUSIONS: PA technology is effective in delivering SHH protein to the penis and SHH is effective in suppressing CN injury-induced apoptosis. These results suggest substantial translational potential of this methodology and show that only a short duration of SHH treatment is required to impact the apoptotic index.