Hemostasis

The fracture model was performed in 3 month-old male mice. Under isoflurane anesthesia a hemostat was used to make a closed fracture of the right tibia just distal to the middle of the tibia. Then the hindlimb was wrapped in casting tape (Delta-Lite) so the hip, knee and ankle were all fixed. The cast extended from the metatarsals of the hindpaw up to a spica formed around the abdomen. A window was left open over the dorsal paw and ankle to prevent constriction when post-fracture edema developed. After fracture and casting, the mice were given subcutaneously 2 days of Buprenorphine (0.05 mg/kg) and Baytril (5 mg/kg) as well as 1.5 ml of normal saline. At 3 weeks after surgery the mice were anesthetized with isoflurane and the cast removed. All mice had union at the fracture site by manual inspection.

The PDE technique utilized in this study employs a gelatin sponge platform (Centenera et al., 2013) (Fig. 1A). This method was selected for two key reasons: firstly, the use of a substrate for explant culture prevents cellular outgrowth that is frequently observed when tissues are cultured without support and completely submerged in media (Pretlow et al., 1995; Varani et al., 1999); secondly, the gelatin sponge used in this study is a commercial medical device developed for hemostasis (Ferrosan, 2014) that is readily available, cost‐effective, and simple to use, making the method feasible for widespread adoption in translational cancer research laboratories.
Figure 1A illustrates the PDE method using prostate tumors as an example. Following surgical tissue removal, the surgeon or clinical pathologist resects a sample of the presumed malignant or nonmalignant region and the specimen is transported to the research laboratory in cold phosphate‐buffered saline on ice, typically within 1–2 h of surgery. Under sterile conditions, tissue was placed onto the lid of a 10‐cm plate along with the saline it was transported in. Using a surgical blade, a 1‐mm‐thick longitudinal section of the tissue sample is cut and placed into neutral‐buffered formalin for paraffin embedding. This tissue is called the Day 0 sample and is used to determine the cancer content of the tissue following staining with hematoxylin and eosin (H&E). The remaining tissue is dissected into 1‐mm³ pieces, called explants, and placed in triplicate or quadruplicate (depending on amount of tissue received) on top of a media‐soaked gelatin sponge (Ethicon; Johnson & Johnson, Somerville, NJ, USA) inside the wells of a 24‐well plate containing 500 μL RPMI 1640 medium containing 10% fetal bovine serum, 1× antimycotic/antibiotic solution, 0.01 mg·mL⁻¹ hydrocortisone, and 0.01 mg·mL⁻¹ insulin. The appropriate vehicle, treatment, or shRNA was added directly to the media inside the appropriate tissue culture well at the indicated concentrations, allowing direct comparison of treatments and controls. Explants were cultured at 37 °C and 5% CO₂ for various time points and then formalin‐fixed and paraffin‐embedded, snap‐frozen, or preserved in RNAlater (Invitrogen, San Diego, CA, USA) depending on the desired downstream analysis. For assessment of BrdU incorporation, BrdU (10 μm) was added to the culture medium 2 h (prostate) or 24 h (breast) prior to harvest.
In our collective experience, all tissues containing epithelial cells can successfully be cultured, and the limiting factor for analysis is instead the presence of sufficient numbers of epithelial (for benign tissue studies) or malignant cells (for cancer tissue studies). For this reason, routine hematoxylin and eosin (H&E) staining of all Day 0 and cultured tissues is an essential part of our protocol to confirm the presence and approximate percentage of benign/malignant cells within the specimens before proceeding with further analyses. Between the three prostate cancer laboratories, our collective experience indicates that approximately 10% of tissues from high‐volume disease and 20–30% of tissues from low‐volume disease do not contain malignant cells, and this is largely due to sampling.

For the PTED group, the surgical procedure (based on the L4–L5 segment of DLS) was performed following methods reported in the literature [18 (link)]. The following steps were performed: (1) part of the superior articular process (SAP) of L5 was removed. A soft pillow was placed under the patients' waist, while the patient was in the lateral decubitus position with their knee and hip flexed. The incision was located 8–12 cm from the midline horizontally and 1–3 cm above the iliac on the side with leg pain. The mixed local anesthetic, which consisted of 30 mL 1:200,000 epinephrine and 20 mL 2% lidocaine, was only used in PTED group. After 5 mL of the mixed anesthetic was inserted into the skin at the entry point, 20 mL was inserted into the trajectory, 15 mL was inserted into the articular process, and 10 mL was inserted into the foramen. Then, 0.8–1.0 cm of skin and the subcutaneous fascia were incised. Drills were used to resect the ventral osteophytes on the SAP. The PTED system (Hoogland Spine Products, Germany) was inserted (Fig. 1). (2) Parts of the ipsilateral ligamentum flavum, perineural scar, and extruded lumbar disc material were completely resected with endoscopic forceps (Fig. 2). (3) The superior endplate of the L5 vertebral body was removed by endoscopic micro punches and a bone knife. Therefore, 270-degree decompression of the traversing nerve root was achieved (Fig. 3). The drainage tube was placed after hemostasis was reached.Fig. 1

Fluoroscopic views. A, B The drill was inserted to resect the LF and the ventral osteophytes on the SAP. C, D The working cannula was placed

Fig. 2

Endoscopic views. A Endoscopic view of the hypertrophic posterior longitudinal ligament, extruded disc material, and perineural scar. B–G After the endoscopic instruments were used to carefully remove the vertebral body, ventral decompression of the traversing nerve root (L5) was completed. H The dura mater was torn

Fig. 3

Illustrations of the 270-degree PTED. A, B Specific pathologic features of LRS-DLS. C, D Final view of the nerve 270-degree decompression status and the restoration of the lateral recess

For the MIS-TLIF group, the surgical procedure was performed in accordance with methods reported in the literature [19 (link)]. After successful general anesthesia with tracheal intubation, the patient was placed in a prone position with chest and hip pads, and the L4–L5 intervertebral space was marked with X-ray fluoroscopy. The skin and subcutaneous fascia were cut; a trans-muscular surgical corridor was created with two micro-laminectomy retractors docking on the facet joint complex. After exposing the bony structure, part of the lamina and inferior articular process of L4 and the upper L5 articular process were removed with the rongeur on the ipsilateral side, and the hypertrophic ligamentum flavum was peeled backward. If MRI showed contralateral lateral recess stenosis, then predecompression was performed on the contralateral side. After decompression on the dorsal side, the nucleus pulposus and endplate cartilage were removed with forceps. An appropriate cage (Medtronic) filled with autograft from laminectomy was placed in the center of the intervertebral space via the Kambin’s triangle area. After adequate hemostasis was achieved, two drainage tubes were placed and removed when the drainage volume was < 50 mL/d.
Postoperatively, patients was treated with oral nonsteroidal anti-inflammatory drugs and antibiotics for 3 days. All patients were encouraged to perform straight leg raising 1 day postoperatively, and moderate off-bed activity with a brace 2–3 days postoperatively. On the third postoperative day, patients were allowed to go home if their lower extremity pain symptoms were effectively relieved with no evidence of infection. The patient demographics and perioperative outcomes were compared. The VAS score, ODI, and modified Macnab criteria were used to evaluate the clinical outcomes [20 (link)].