Reconstruction of large and complex hard tissue defects remains a major clinical challenge, as conventional autografts and allografts are often limited in availability, biological compatibility, and long-term efficacy, particularly for extensive defects or poor bone quality. Recombinant human bone morphogenetic protein-2 (rhBMP-2) is a potent osteoinductive factor capable of initiating the complete cascade of bone formation. However, its clinical use is restricted by dose-dependent complications such as inflammation, ectopic ossification, and osteolysis. This review synthesizes current evidence on the safety profile of rhBMP-2 and examines strategies to enhance its therapeutic index. Preclinical and clinical data indicate that conventional collagen-based carriers frequently cause rapid burst release and uncontrolled diffusion, aggravating adverse outcomes. It is noteworthy that low doses of rhBMP-2 (0.5–0.7 mg/level in anterior cervical discectomy and fusion (ACDF) or 0.5–1.0 mg/level in transforaminal lumbar interbody fusion (TLIF)) provide the optimal balance of efficacy and safety. Advanced biomaterial-based platforms, such as bioceramic–polymer composites, injectable hydrogels, and 3D-printed scaffolds, enable spatially and temporally controlled release while maintaining osteogenic efficacy. Molecular delivery approaches, including chemically modified messenger RNA (cmRNA) and regional gene therapy, provide transient, site-specific rhBMP-2 expression with reduced dosing and minimal systemic exposure. By integrating mechanistic insights with translational advances, this review outlines a framework for optimizing rhBMP-2-based regenerative protocols, emphasizing their potential role in multidisciplinary strategies for reconstructing complex hard tissue defects.

Isolation of pancreatic islets for single-cell RNA sequencing (scRNA-seq) remains technically challenging because enzymatic digestion, mechanical dissociation, and subsequent purification can cause cell loss, aggregation, and processing-induced transcriptional changes. This study aimed to adapt a practical gradient-free alternative for preparing murine pancreatic islets for downstream single-cell transcriptomic analysis while reducing the number of sample-processing steps. The workflow combined intraductal perfusion of the pancreas with 3 mL of a collagenase solution through the common bile duct, controlled enzymatic digestion at 37 °C, performed manual islet selection without density-gradient purification, and achieved gentle dissociation with Accutase and EDTA. DNase I was included in all post-digestion steps to limit cell aggregation. The protocol was evaluated using pancreatic islets from BKS.Cg-Dock7^m +/+ Lepr^db/J mice, a model of type 2 diabetes. Compared with Ficoll-based purification, the gradient-free protocol provided viable cell yields within a similar range while eliminating density-gradient preparation, an 18 min gradient centrifugation step, two subsequent wash centrifugations, and the associated transfer and dilution steps. The resulting scRNA-seq library showed satisfactory quality-control (QC) characteristics and retained the major endocrine populations, including β-, α-, δ-, and PP cells. The gradient-free sample also showed a lower overall stress-response score and lower expression of several immediate early genes, although heat shock- and endoplasmic reticulum (ER) stress-associated genes displayed heterogeneous patterns. These differences were considered descriptive because only one pooled scRNA-seq library was generated for each workflow. These findings support gradient-free isolation as a possible alternative to Ficoll-based purification for preparing murine pancreatic islets for scRNA-seq analysis.