Durable immunomodulatory hierarchical patch for rotator cuff repairing.

Degradable rotator cuff patches, followed over five years, have been observed to exhibit high re-tear rates exceeding 50%, which is attributed to the inability of degradable polymers alone to restore the post-rotator cuff tear (RCT) inflammatory niche. Herein, poly(ester-ferulic acid-urethane)urea (PEFUU) was developed, featuring prolonged anti-inflammatory functionality, achieved by the integration of ferulic acid (FA) into the polyurethane repeating units. PEFUU stably releases FA in vitro, reversing the inflammatory niche produced by M1 macrophages and restoring the directed differentiation of stem cells. Utilizing PEFUU, hierarchical composite nanofiber patch (HCNP) was fabricated, simulating the natural microstructure of the tendon-to-bone interface with an aligned-random alignment. The incorporation of enzymatic hydrolysate derived from decellularized Wharton jelly tissue into the random layer could further enhance cartilage regeneration at the tendon-to-bone interface. Via rat RCT repairing model, HCNP possessing prolonged anti-inflammatory properties uniquely facilitated physiological healing at the tendon-to-bone interface's microstructure. The alignment of fibers was restored, and histologically, the characteristic tripartite distribution of collagen I - collagen II - collagen I was achieved. This study offers a universal approach to the functionalization of degradable polymers and provides a foundational reference for their future applications in promoting the in vivo regeneration of musculoskeletal tissues.

Efficient expansion and CRISPR-Cas9-mediated gene correction of patient-derived hepatocytes for treatment of inherited liver diseases.

Cell-based ex vivo gene therapy in solid organs, especially the liver, has proven technically challenging. Here, we report a feasible strategy for the clinical application of hepatocyte therapy. We first generated high-quality autologous hepatocytes through the large-scale expansion of patient-derived hepatocytes. Moreover, the proliferating patient-derived hepatocytes, together with the AAV2.7m8 variant identified through screening, enabled CRISPR-Cas9-mediated targeted integration efficiently, achieving functional correction of pathogenic mutations in FAH or OTC. Importantly, these edited hepatocytes repopulated the injured mouse liver at high repopulation levels and underwent maturation, successfully treating mice with tyrosinemia following transplantation. Our study combines ex vivo large-scale cell expansion and gene editing in patient-derived transplantable hepatocytes, which holds potential for treating human liver diseases.

Engineering APOBEC3A deaminase for highly accurate and efficient base editing.

Cytosine base editors (CBEs) are effective tools for introducing C-to-T base conversions, but their clinical applications are limited by off-target and bystander effects. Through structure-guided engineering of human APOBEC3A (A3A) deaminase, we developed highly accurate A3A-CBE (haA3A-CBE) variants that efficiently generate C-to-T conversion with a narrow editing window and near-background level of DNA and RNA off-target activity, irrespective of methylation status and sequence context. The engineered deaminase domains are compatible with PAM-relaxed SpCas9-NG variant, enabling accurate correction of pathogenic mutations in homopolymeric cytosine sites through flexible positioning of the single-guide RNAs. Dual adeno-associated virus delivery of one haA3A-CBE variant to a mouse model of tyrosinemia induced up to 58.1% editing in liver tissues with minimal bystander editing, which was further reduced through single dose of lipid nanoparticle-based messenger RNA delivery of haA3A-CBEs. These results highlight the tremendous promise of haA3A-CBEs for precise genome editing to treat human diseases.

Sacubitril/valsartan inhibits the proliferation of vascular smooth muscle cells through notch signaling and ERK1/2 pathway.

AIMS: To explore the role and mechanism of Notch signaling and ERK1/2 pathway in the inhibitory effect of sacubitril/valsartan on the proliferation of vascular smooth muscle cells (VSMCs). MAIN METHODS: Human aortic vascular smooth muscle cells (HA-VSMCs) were cultured in vitro. The proliferating VSMCs were divided into three groups as control group, Ang II group and Ang II + sacubitril/valsartan group. Cell proliferation and migration were detected by CCK8 and scratch test respectively. The mRNA and protein expression of PCNA, MMP-9, Notch1 and Jagged-1 were detected by qRT-PCR and Western blot respectively. The p-ERK1/2 expression was detected by Western blot. KEY FINDINGS: Compared with the control group, proliferation and migration of VSMCs and the expression of PCNA, MMP-9, Notch1, Jagged-1 and p-ERK1/2 was increased in Ang II group. Sacubitril/valsartan significantly reduced the proliferation and migration. Additionally, pretreatment with sacubitril/valsartan reduced the PCNA, MMP-9, Notch1, Jagged-1 and p-ERK1/2 expression.

The non-causative role of abnormal serum uric acid in intervertebral disc degeneration: A Mendelian randomization study.

Background: Intervertebral disc degeneration (IDD) is a common musculoskeletal disorder that contributes significantly to disability and healthcare costs. Serum urate concentration has been implicated in the development of various musculoskeletal conditions. While previous observational studies have suggested an association between the two conditions, it might confound the effect of serum urate concentrations on IDD. This Mendelian randomization (MR) study aimed to investigate the causal relationship between serum urate concentration and IDD. Methods: We performed a two-sample MR analysis using summary-level data from genome-wide association studies (GWAS) of serum urate concentration (n = 13 585 994 European ancestry) and IDD (n = 16 380 337 European ancestry). Single nucleotide polymorphisms (SNPs) significantly associated with serum urate concentration (p < 5 × 10-8) were selected as instrumental variables. The associations between genetically predicted serum urate concentration and IDD were estimated using the inverse-variance weighted (IVW) method, with sensitivity analyses employing the weighted median, MR-Egger, and MR-PRESSO approaches to assess the robustness of the findings. Results: In the primary IVW analysis, genetically predicted serum urate concentration was unrelated associated with IDD (odds ratio [OR] = 1.00, 95% confidence interval (CI): 1.00-1.00, p = 0.17)). The results remained consistent across the sensitivity analyses, and no significant directional pleiotropy was detected (MR-Egger intercept: p = 0.15). Conclusions: This MR study provides evidence that there is no causal relationship between serum urate concentration and IDD. It suggests previous observational associations may be confounded. Serum urate levels are unlikely to be an important contributor to IDD.

Adenine transversion editors enable precise, efficient A•T-to-C•G base editing in mammalian cells and embryos.

Base editors have substantial promise in basic research and as therapeutic agents for the correction of pathogenic mutations. The development of adenine transversion editors has posed a particular challenge. Here we report a class of base editors that enable efficient adenine transversion, including precise A•T-to-C•G editing. We found that a fusion of mouse alkyladenine DNA glycosylase (mAAG) with nickase Cas9 and deaminase TadA-8e catalyzed adenosine transversion in specific sequence contexts. Laboratory evolution of mAAG significantly increased A-to-C/T conversion efficiency up to 73% and expanded the targeting scope. Further engineering yielded adenine-to-cytosine base editors (ACBEs), including a high-accuracy ACBE-Q variant, that precisely install A-to-C transversions with minimal Cas9-independent off-targeting effects. ACBEs mediated high-efficiency installation or correction of five pathogenic mutations in mouse embryos and human cell lines. Founder mice showed 44-56% average A-to-C edits and allelic frequencies of up to 100%. Adenosine transversion editors substantially expand the capabilities and possible applications of base editing technology.

Decellularization-Based Modification Strategy for Bioactive Xenografts Promoting Tendon Repair.

Xenografts have emerged as a promising option for severe tendon defects treatment. However, despite undergoing decellularization, concerns still remain regarding the immunogenicity of xenografts. Because certain components within the extracellular matrix also possess immunogenicity. In this study, a novel strategy of post-decellularization modification aimed at preserving the endogenous capacity of cells on collagen synthesis to mask antigenic epitopes in extracellular matrix is proposed. To implement this strategy, a human-derived rosiglitazone-loaded decellularized extracellular matrix (R-dECM) is developed. R-dECM can release rosiglitazone for over 7 days in vitro. By suppressing M1 macrophage polarization, R-dECM protects the migration and collagen synthesis abilities of tendon-derived stem cells (TDSCs), while also stabilizing the phenotype of M2 macrophages in vitro. RNA sequencing reveals R-dECM can mitigate the detrimental crosstalk between TDSCs and inflammatory cells. When applied to a rat patellar tendon defect model, R-dECM effectively inhibits early inflammation, preventing chronic inflammation. Its duration of function far exceeds the release time of rosiglitazone, implying the establishment of immune evasion, confirming the effectiveness of the proposed strategy. And R-dECM demonstrates superior tendon repair outcomes compared to dECM. Thus, this study provides a novel bioactive scaffold with the potential to enhance the long-term clinical outcomes of xenogeneic tendon grafts.

Durable Immunomodulatory Nanofiber Niche for the Functional Remodeling of Cardiovascular Tissue.

Functional remodeling and prolonged anti-inflammatory responses are both vital for repairing damage in the cardiovascular system. Although these aspects have each been studied extensively alone, attempts to fabricate scaffolds that combine these effects have seen limited success. In this study, we synthesized salvianic acid A (SA, danshensu) blocked biodegradable polyurethane (PCHU-D) and enclosed it within electrospun nanofibers to synthesize a durable immunomodulatory nanofiber niche (DINN), which provided sustained SA release during inflammation. Given its excellent processability, mechanical properties, and shape memory function, we developed two variants of the DINN as vascular scaffolds and heart patches. Both these variants exhibited outstanding therapeutic effects in in vivo experiments. The DINN was expertly designed such that it gradually decomposes along with SA release, substantially facilitating cellular infiltration and tissue remodeling. Therefore, the DINN effectively inhibited the migration and chemotaxis of inflammatory cells, while also increasing the expression of angiogenic genes. As a result, it promoted the recovery of myocardial function after myocardial infarction and induced rapid reendothelialization following arterial orthotopic transplantation repair. These excellent characteristics indicate that the DINN holds great potential as a multifunctional agent for repairing cardiovascular tissues.

Bi-lineage inducible and immunoregulatory electrospun fibers scaffolds for synchronous regeneration of tendon-to-bone interface.

Facilitating regeneration of the tendon-to-bone interface can reduce the risk of postoperative retear after rotator cuff repair. Unfortunately, undesirable inflammatory responses following injury, difficulties in fibrocartilage regeneration, and bone loss in the surrounding area are major contributors to suboptimal tendon-bone healing. Thus, the development of biomaterials capable of regulating macrophage polarization to a favorable phenotype and promoting the synchronous regeneration of the tendon-to-bone interface is currently a top priority. Here, strontium-doped mesoporous bioglass nanoparticles (Sr-MBG) were synthesized through a modulated sol-gel method and Bi-lineage Inducible and Immunoregulatory Electrospun Fibers Scaffolds (BIIEFS) containing Sr-MBG were fabricated. The BIIEFS were biocompatible, showed sustained release of multiple types of bioactive ions, enhanced osteogenic and chondrogenic differentiation of mesenchymal stem cells (MSCs), and facilitated macrophage polarization towards the M2 phenotype in vitro. The implantation of BIIEFS at the torn rotator cuff resulted in greater numbers of M2 macrophages and the synchronous regeneration of tendon, fibrocartilage, and bone at the tendon-to-bone interface, leading to a significant improvement in the biomechanical strength of the supraspinatus tendon-humerus complexes. Our research offers a feasible strategy to fabricate immunoregulatory and multi-lineage inducible electrospun fibers scaffolds incorporating bioglass nanoparticles for the regeneration of soft-to-hard tissue interfaces.

Feedback activation of EGFR/wild-type RAS signaling axis limits KRASG12D inhibitor efficacy in KRASG12D-mutated colorectal cancer.

Colorectal cancer (CRC), which shows a high degree of heterogeneity, is the third most deadly cancer worldwide. Mutational activation of KRASG12D occurs in approximately 10-12% of CRC cases, but the susceptibility of KRASG12D-mutated CRC to the recently discovered KRASG12D inhibitor MRTX1133 has not been fully defined. Here, we report that MRTX1133 treatment caused reversible growth arrest in KRASG12D-mutated CRC cells, accompanied by partial reactivation of RAS effector signaling. Through a drug-anchored synthetic lethality screen, we discovered that epidermal growth factor receptor (EGFR) inhibition was synthetic lethal with MRTX1133. Mechanistically, MRTX1133 treatment downregulated the expression of ERBB receptor feedback inhibitor 1 (ERRFI1), a crucial negative regulator of EGFR, thereby causing EGFR feedback activation. Notably, wild-type isoforms of RAS, including H-RAS and N-RAS, but not oncogenic K-RAS, mediated signaling downstream of activated EGFR, leading to RAS effector signaling rebound and reduced MRTX1133 efficacy. Blockade of activated EGFR with clinically used antibodies or kinase inhibitors suppressed the EGFR/wild-type RAS signaling axis, sensitized MRTX1133 monotherapy, and caused the regression of KRASG12D-mutant CRC organoids and cell line-derived xenografts. Overall, this study uncovers feedback activation of EGFR as a prominent molecular event that restricts KRASG12D inhibitor efficacy and establishes a potential combination therapy consisting of KRASG12D and EGFR inhibitors for patients with KRASG12D-mutated CRC.

Improving adenine and dual base editors through introduction of TadA-8e and Rad51DBD.

Base editors, including dual base editors, are innovative techniques for efficient base conversions in genomic DNA. However, the low efficiency of A-to-G base conversion at positions proximal to the protospacer adjacent motif (PAM) and the A/C simultaneous conversion of the dual base editor hinder their broad applications. In this study, through fusion of ABE8e with Rad51 DNA-binding domain, we generate a hyperactive ABE (hyABE) which offers improved A-to-G editing efficiency at the region (A10-A15) proximal to the PAM, with 1.2- to 7-fold improvement compared to ABE8e. Similarly, we develop optimized dual base editors (eA&C-BEmax and hyA&C-BEmax) with markedly improved simultaneous A/C conversion efficiency (1.2-fold and 1.5-fold improvement, respectively) compared to A&C-BEmax in human cells. Moreover, these optimized base editors catalyze efficiently nucleotide conversions in zebrafish embryos to mirror human syndrome or in human cells to potentially treat genetic diseases, indicating their great potential in broad applications for disease modeling and gene therapy.

Exosomes Derived From Kartogenin-Preconditioned Mesenchymal Stem Cells Promote Cartilage Formation and Collagen Maturation for Enthesis Regeneration in a Rat Model of Chronic Rotator Cuff Tear.

BACKGROUND: Poor tendon-to-bone healing in chronic rotator cuff tears (RCTs) is related to unsatisfactory outcomes. Exosomes derived from mesenchymal stem cells reportedly enhance rotator cuff healing. However, the difficulty in producing exosomes with a stronger effect on enthesis regeneration must be resolved. PURPOSE: To study the effect of exosomes derived from kartogenin (KGN)-preconditioned human bone marrow mesenchymal stem cells (KGN-Exos) on tendon-to-bone healing in a rat model of chronic RCT. STUDY DESIGN: Controlled laboratory study. METHODS: Exosome-loaded sodium alginate hydrogel (SAH) was prepared. Moreover, exosomes were labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) or 1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate (Dil) for in vivo tracking. Bilateral rotator cuff repair (RCR) was conducted in an established chronic RCT rat model. A total of 66 rats were randomized to control, untreated exosome (un-Exos), and KGN-Exos groups to receive local injections of pure SAH, un-Exos, or KGN-Exos SAH at the repaired site. The presence of DiR/Dil-labeled exosomes was assessed at 1 day and 1 week, and tendon-to-bone healing was evaluated histologically, immunohistochemically, and biomechanically at 4 and 8 weeks. RESULTS: Both un-Exos and KGN-Exos exhibited sustained release from SAH for up to 96 hours. In vivo study revealed that un-Exos and KGN-Exos were localized to the repaired site at 1 week. Moreover, the KGN-Exos group showed a higher histological score and increased glycosaminoglycan and collagen II expression at 4 and 8 weeks. In addition, more mature and better-organized collagen fibers with higher ratios of collagen I to collagen III were observed at 8 weeks in the tendon-to-bone interface compared with those in the control and un-Exos groups. Biomechanically, the KGN-Exos group had the highest failure load (28.12 ± 2.40 N) and stiffness (28.57 ± 2.49 N/mm) among the 3 groups at 8 weeks. CONCLUSION: Local injection of SAH with sustained KGN-Exos release could effectively promote cartilage formation as well as collagen maturation and organization for enthesis regeneration, contributing to enhanced biomechanical properties after RCR. CLINICAL RELEVANCE: KGN-Exos injection may be used as a cell-free therapeutic option to accelerate tendon-to-bone healing in chronic RCT.

Generation of genetically modified rat models via the CRISPR/Cas9 technology.

The RNA-guided CRISPR/Cas9 genomic editing system consists of a single guide RNA (sgRNA) and a Cas9 nuclease. The two components form a complex in cells and target the genomic loci complementary to the sgRNA. The Cas9 nuclease cleaves the target site creating a double stranded DNA break (DSB). In mammalian cells, DSBs are often repaired via error prone non-homologous end joining (NHEJ) or via homology directed repair (HDR) with the presence of donor DNA templates. Micro-injection of the CRISPR/Cas9 system into the rat embryos enables generation of genetically modified rat models. Here, we describe a detailed protocol for creating gene knockout or knockin rat models via the CRISPR/Cas9 technology.

Arthroscopic Dynamic Anterior Stabilization Using Either Long Head of the Biceps or Conjoined Tendon Transfer for Anterior Shoulder Instability Results in a Similarly Low Recurrence Rate.

PURPOSE: To compare the clinical outcomes of arthroscopic dynamic anterior stabilization (DAS) between transferring the long head of the biceps (DAS-LHB) and the conjoined tendon (DAS-CT) for anterior shoulder instability with <15% glenoid bone loss. METHODS: From January 2016 to May 2019, a total of 63 patients who underwent DAS for recurrent anterior shoulder dislocation with <15% glenoid bone loss were included, comprising 33 patients in DAS-LHB group and 30 patients in DAS-CT group. Clinical outcomes were assessed preoperatively and at a minimum 3-year follow-up, including patient-reported outcomes, range of motion, and return to sports (RTS). Postoperative recurrent instability (including dislocation, subluxation, and subjective instability with a positive apprehension test), revisions and complications also were recorded. RESULTS: No significant demographic characteristics difference was detected between the DAS-LHB (26.3 ± 7.9 years) and DAS-CT groups (26.0 ± 6.7 years). At the latest follow-up, there were no significant differences between the 2 groups in functional scores: Oxford Shoulder Instability Score (14.8 ± 2.8 vs 15.2 ± 3.6), Rowe score (95.9 ± 6.5 vs 93.2 ± 10.2), visual analog scale for pain (0.8 ± 1.2 vs 0.7 ± 1.7), and American Shoulder and Elbow Surgeons (95 ± 8.8 vs 95.2 ± 9.1) (all P > .218). No significant difference was detected between groups in the rates of RTS (90.1% vs 86.7%, P = .700) and RTS at previous level (78.7% vs 73.3%, P = .258), respectively. No recurrent dislocation occurred in either group. One patient felt occasional subluxation in the DAS-LHB group, and one was positive for the apprehension test in each group. One patient presented with postoperative shoulder stiffness and underwent a secondary arthroscopic debridement in the DAS-CT group. CONCLUSIONS: Comparable rates of recurrence, complication, return to sports, and subjective shoulder function were observed between DAS-LHB and DAS-CT groups. LEVEL OF EVIDENCE: Level III, retrospective comparative therapeutic trial.

Engineering a precise adenine base editor with minimal bystander editing.

Adenine base editors (ABEs) catalyze A-to-G transitions showing broad applications, but their bystander mutations and off-target editing effects raise safety concerns. Through structure-guided engineering, we found ABE8e with an N108Q mutation reduced both adenine and cytosine bystander editing, and introduction of an additional L145T mutation (ABE9), further refined the editing window to 1-2 nucleotides with eliminated cytosine editing. Importantly, ABE9 induced very minimal RNA and undetectable Cas9-independent DNA off-target effects, which mainly installed desired single A-to-G conversion in mouse and rat embryos to efficiently generate disease models. Moreover, ABE9 accurately edited the A5 position of the protospacer sequence in pathogenic homopolymeric adenosine sites (up to 342.5-fold precision over ABE8e) and was further confirmed through a library of guide RNA-target sequence pairs. Owing to the minimized editing window, ABE9 could further broaden the targeting scope for precise correction of pathogenic single-nucleotide variants when fused to Cas9 variants with expanded protospacer adjacent motif compatibility. bpNLS, bipartite nuclear localization signals.

Clinical and Radiological Outcomes in Patients With Anterior Shoulder Instability and Glenoid Bone Loss after Arthroscopic Free Bone Block Combined With Dynamic Anterior Stabilization.

BACKGROUND: As an alternative to the Latarjet procedure, the arthroscopic free bone block (FBB) procedure combined with dynamic anterior stabilization (DAS) has been recently proposed to provide both glenoid augmentation and a tendon sling effect for treating anterior shoulder instability (ASI) with glenoid bone loss. PURPOSE: To evaluate the clinical and radiological outcomes of FBB-DAS for ASI with glenoid bone loss. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: Patients who underwent arthroscopic FBB-DAS for ASI with >15% glenoid bone loss between February 2017 and March 2020 were screened and enrolled in this study. Clinical outcome measures were assessed preoperatively and at a minimum 2-year follow-up, including recurrence, complications, shoulder functional scores, range of motion, and return to sports. Postoperative computed tomography and magnetic resonance imaging were also performed. RESULTS: Of a total of 65 patients with a mean follow-up of 46.1 ± 13.1 months, no patients experienced a recurrent dislocation or subluxation postoperatively, while 2 had a positive anterior apprehension test (3.1%). Additionally, 2 patients (3.1%) experienced complications of hematoma and shoulder stiffness, respectively. The mean visual analog scale score, American Shoulder and Elbow Surgeons score, Rowe score, and Oxford Shoulder Instability Score all improved significantly from 3.2 ± 2.4, 75.0 ± 18.9, 43.6 ± 27.3, and 33.8 ± 9.0 preoperatively to 1.3 ± 0.8, 95.1 ± 8.0, 95.5 ± 7.8, and 14.8 ± 3.5 at final follow-up, respectively (all P < .001). No difference was detected in range of motion except for 8.1° and 7.5° external rotation limitations in adduction and abduction, respectively. There were 62 patients (95.4%) who returned to sports, and 54 patients (83.1%) returned to the preinjury level. The transferred biceps tendon was intact in all 59 patients who completed radiological examination at the latest follow-up. Good bone healing was achieved in 98.3% of patients, and the glenoid bone defect decreased from 18.1% to 4.9%. Osseous and labral glenoids were significantly enlarged in width and depth on the latest magnetic resonance imaging (all P < .001). CONCLUSION: Arthroscopic FBB-DAS provided satisfactory clinical and radiological outcomes for ASI with glenoid bone loss. Despite slight external rotation restrictions, it achieved low recurrence and complication rates, excellent shoulder functional scores, a high return-to-sports rate, and favorable graft healing and remodeling.

Engineering of efficiency-enhanced Cas9 and base editors with improved gene therapy efficacies.

Editing efficiency is pivotal for the efficacies of CRISPR-based gene therapies. We found that fusing an HMG-D domain to the N terminus of SpCas9 (named efficiency-enhanced Cas9 [eeCas9]) significantly increased editing efficiency by 1.4-fold on average. The HMG-D domain also enhanced the activities of non-NGG PAM Cas9 variants, high-fidelity Cas9 variants, smaller Cas9 orthologs, Cas9-based epigenetic regulators, and base editors in cell lines. Furthermore, we discovered that eeCas9 exhibits comparable off-targeting effects with Cas9, and its specificity could be increased through ribonucleoprotein delivery or using hairpin single-guide RNAs and high-fidelity Cas9s. The entire eeCas9 could be packaged into an adeno-associated virus vector and exhibited a 1.7- to 2.6-fold increase in editing efficiency targeting the Pcsk9 gene in mice, leading to a greater reduction of serum cholesterol levels. Moreover, the efficiency of eeA3A-BE3 also surpasses that of A3A-BE3 in targeting the promoter region of γ-globin genes or BCL11A enhancer in human hematopoietic stem cells to reactivate γ-globin expression for the treatment of ß-hemoglobinopathy. Together, eeCas9 and its derivatives are promising editing tools that exhibit higher activity and therapeutic efficacy for both in vivo and ex vivo therapeutics.