Stem Cell–Conditioned Medium Promotes Graft Remodeling of Midsubstance and Intratunnel Incorporation After Anterior Cruciate Ligament Reconstruction in a Rat Model

Abstract

Background: Stem cell–conditioned medium (CM) has been increasingly used in regenerative medicine. However, its effect on graft-host integration after anterior cruciate ligament (ACL) reconstruction (ACLR) remains unclear. Purpose: To examine the effect of human bone marrow stem cell (hBMSC)–CM on graft-bone integration and graft midsubstance ligamentization in a rat model of ACLR. Study Design: Controlled laboratory study. Methods: CM was obtained from the supernatant of commercially available hBMSCs in serum-free Dulbecco’s modified Eagle medium (DMEM). In a rat model of an ACL injury, isometric ACLR was performed. Three groups were established: CM injection group (CM; n = 40), control injection group (CI; n = 40) with serum-free DMEM injections, and no injection group (NI; n = 40). An intra-articular injection was performed weekly. Micro–computed tomography was conducted at 2, 4, and 8 weeks postoperatively. Histological and biomechanical analyses were conducted at 4 and 8 weeks postoperatively. The NIH3T3 fibroblast was utilized as a model in vitro to examine the effect of CM using the cell counting kit–8 (CCK-8) assay and immunofluorescence staining of Ki-67, α–smooth muscle actin (α-SMA), and collagen 1 (Col 1). Results: At 4 and 8 weeks, the femoral and tibial bone tunnel areas as well as the interface between the graft and host bone were smaller, while the bone volume/total volume ratio was higher, in the CM group. Sharpey-like fibers formed at 8 weeks in the CM group. At 4 and 8 weeks, more Col 1 was noticed in the CM group than in the NI group (both P < .001) or CI group (both P < .001). Immunohistochemically, the α-SMA–positive area was up-regulated at the graft-bone interface at 4 weeks ( P < .001) and declined at 8 weeks ( P < .001) in the CM group compared with the other 2 groups. At the midsubstance, α-SMA expression decreased from 4 to 8 weeks in all groups and was significantly lower in the CM group than in the NI group ( P < .01) or CI group ( P < .05) at 8 weeks. The CCK-8 assay showed that CM increased NIH3T3 viability ( P < .001) and the level of Ki-67 ( P < .05), α-SMA ( P < .001), and Col 1 ( P < .001) in CM-educated NIH3T3 cells. Conclusion: hBMSC-CM accelerates graft-bone incorporation and midsubstance ligamentization and enhances the proliferation, differentiation, and collagen synthesis of fibroblasts. Clinical Relevance: Graft-host integration is essential after ACLR. The current study identified a novel agent, that is, hBMSC-CM, as a candidate for promoting integration.

An Autograft for Anterior Cruciate Ligament Reconstruction Results in Better Biomechanical Performance and Tendon-Bone Incorporation Than Does a Hybrid Graft in a Rat Model

“…4,7,8,47 The mean interface width of a sample was calculated by averaging the width at every 30° point of the tunnel cross section from 0° to 360°, and 3 sections were measured per sample. 47 The graft-bone integration parameters were analyzed using a previously validated scoring system. 28 This scoring system was based on a scaled maximum of 20 points with a range from 0 to 4 points in each parameter.…”

“…The tendon graft was then passed through the tunnels. The knee was next brought to 30°of flexion, at which point the graft was pretensioned to 5 N. 29 Both ends of the tendon graft were sutured to the surrounding periosteum at the exit of the tunnel using 4-0/T nonabsorbable suture 29,47,48 ( Figure 2C). The wounds were closed with sutures, and physical activity was not restricted postoperatively.…”

Zanthoxylum nitidum extract attenuates BMP-2-induced inflammation and hyperpermeability

“…To examine the expression and location of p65 and VE-Cadherin, Immunofluorescence staining on HUVECs was conducted as previously described [ 27 ]. Briefly, cells were fixed with 4% paraformaldehyde about 8 h and then blocked with donkey serum for 30 min.…”

The Serum from Patients with Secondary Frozen Shoulder Following Rotator Cuff Repair Induces Shoulder Capsule Fibrosis and Promotes Macrophage Polarization and Fibroblast Activation

“…HE and Masson staining were conducted based on the established method. 34,35 The thickness of joint capsule was obtained by five random measurements to obtain a mean value using ImageJ software. The relative thickness of joint capsule in different groups compared to group NC was calculated.…”