Impact of age and donor sites on bioactivities of tendon cells in autologous tenocyte implantation (OrthoATI™) for treatment of chronic tendinopathy.

OBJECTIVES: Autologous tenocyte implantation (OrthoATI™) therapy has demonstrated efficacy in treating patients with tendinopathy at various anatomical sites. This study evaluates the effect of patient age, gender, and tendon biopsy site on morphology, growth, and gene expression of autologous tendon cells used to treat chronic tendinopathy. METHODS: Patients undergoing OrthoATI™ for tendinopathies between 2020 and 2022 were initially treated by biopsies taken from patella tendon (PT) or palmaris longus tendon (PL). The biopsies were sent to a Good Manufacturing Practice (GMP) cell laboratory where tendon cells were isolated, cultured, and expanded for four to six weeks. Cell morphology was assessed using phase contrast microscopy. Droplet digital PCR (ddPCR) was utilized for gene expression analysis. Dichotomous results were compared between groups using x2 or Fisher's exact tests with no adjustment for multiple comparisons. The nonparametric Mann-Whitney U and Kruskal-Wallis tests were utilized for the sex and age (<35y, 35-44y, 45-54y, >55y) analyses, respectively. All analyses were performed using IBM SPSS v27, and a two-tailed P-value of <0.05 was considered statistically significant. RESULTS: 149 patients were included in the analysis. The PT was biopsied in 63 patients, and PL in 86 patients. There were no observer effects for age and gender between the PT and PL groups. There was no statistical significance between the PT and PL tendons for cell morphology, average cell population doubling time (PDT) (PT 83.9 vs PL 82.7 â€‹h, p â€‹= â€‹0.482), cellular yield (PT 16.2 vs PL 15.2 â€‹× â€‹106, p â€‹= â€‹0.099), and cell viability (PT 98.7 vs PL 99.0%, p â€‹= â€‹0.277). Additionally, ddPCR analyses showed no statistical significance found in tenogenic gene expression, including collagen type I (COL1, p â€‹= â€‹0.86), tenomodulin (TNMD, p â€‹= â€‹0.837) and scleraxis (SCX, p â€‹= â€‹0.331) between PT- and PL-derived tendon cells. An age stratification analysis found no effect on growth and gene expression. COL1 was found to be higher in males when compared to females (P â€‹< â€‹0.001), but otherwise no difference was seen in growth and gene expression in the gender analysis. No postbiopsy clinical complications were reported for either group. CONCLUSION: This study has shown that the growth and bioactivities of tendon cells from tendon biopsies for OrthoATI™ are not affected by tendon donor site and age. LEVEL OF EVIDENCE: IV.

A bio-inductive collagen scaffold that supports human primary tendon-derived cell growth for rotator cuff repair.

BACKGROUND: Rotator Cuff (RC) tendon tearing is a common clinical problem and there is a high incidence of revision surgery due to re-tearing. In an effort to improve patient outcome and reduce surgical revision, scaffolds have been widely used for augmentation of RC repairs. However, little is known about how scaffolds support tendon stem cell growth or facilitate tendon regeneration. The purpose of this study is to evaluate the structural and biological properties of a bioactive collagen scaffold (BCS) with the potential to promote tendon repair. Additionally, we conducted a pilot clinical study to assess the safety and feasibility of using the BCS for repair of RC tears. METHODS: A series of physical, ultrastructural, molecular and in vitro tests determined the biocompatibility and teno-inductive properties of this BCS. In addition, a prospective case study of 18 patients with RC tendon tears (>20 â€‹mm in diameter) was performed in an open-label, single-arm study, involving either mini-open or arthroscopic surgical RC repair with the BCS. Clinical assessment of RC repair status was undertaken by MRI-imaging at baseline, 6 and 12 months and patient evaluated questionnaires were taken at baseline as well as 3, 6 & 12 months. RESULTS: The BCS consists of highly purified type-I collagen, in bundles of varying diameter, arranged in a higher order tri-laminar structure. BCS have minimal immunogenicity, being cell and essentially DNA-free as well as uniformly negative for the porcine α-Gal protein. BCS seeded with human primary tendon-derived cells and exposed to 6% uniaxial loading conditions in vitro, supported increased levels of growth and proliferation as well as up-regulating expression of tenocyte differentiation marker genes including TNMD, Ten-C, Mohawk and Collagen-1α1. To test the safety and feasibility of using the BCS for augmentation of RC repairs, we followed the IDEAL framework and conducted a first, open-label single arm prospective case series study of 18 patients. One patient was withdrawn from the study at 3 months due to wound infection unrelated to the BCS. The remaining 17 cases showed that the BCS is safe to be implanted. The patients reported encouraging improvements in functional outcomes (ASES, OSS and Constant-Murley scores), as well as quality of life assessments (AQoL) and a reduction in VAS pain scores. MRI assessment at 12 months revealed complete healing in 64.8% patients (11/17), 3 partial thickness re-tears (17.6%) and 3 full thickness re-tears (17.6%). CONCLUSION: The BCS is composed of type-I collagen that is free of immunogenic proteins and supports tendon-derived cell growth under mechanical loading in vitro. This pilot study shows that it is safe and feasible to use BCS for RC argumentation and further controlled prospective studies are required to demonstrate its efficacy. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The results of this study indicate that this bioactive collagen scaffold has unique properties for supporting tendon growth and that it is non-immunogenic. The clinical study further confirms that the scaffold is a promising biological device for augment of human rotator cuff repairs.

Collagen Membrane for Guided Bone Regeneration in Dental and Orthopedic Applications.

Treatment of cortical bone defects is a clinical challenge. Guided bone regeneration (GBR), commonly used in oral and maxillofacial dental surgery, may show promise for orthopedic applications in repair of cortical bone defects. However, a limitation in the use of GBR for cortical bone defects is the lack of an ideal scaffold that provides sufficient mechanical support to bridge the cortical bone with minimal interference in the repair process. We have developed a new collagen membrane, CelGro™, for use in GBR. We report the material characterization of CelGro and evaluate the performance of CelGro in translational preclinical and clinical studies. The results show CelGro has a bilayer structure of different fiber alignment and is composed almost exclusively of type I collagen. CelGro was found to be completely acellular and free from xenoantigen, α-gal (galactose-alpha-1,3-galactose). In the preclinical study of a rabbit cortical bone defect model, CelGro demonstrated enhanced bone-remodeling activity and cortical bone healing. Microcomputed tomography evaluation showed early bony bridging over the defect area 30 days postoperatively, and nearly complete restoration of mature cortical bone at the bone defect site 60 days postoperatively. Histological analysis 60 days after surgery further confirmed that CelGro enables bridging of the cortical bone defect by induction of newly formed cortical bone. Compared to a commercially available collagen membrane, Bio-Gide®, CelGro showed much better cortical alignment and reduced porosity at the defect interface. As selection of orthopedic patients with cortical bone defects is complex, we conducted a clinical study evaluating the performance of CelGro in guided bone regeneration around dental implants. CelGro was used in GBR procedures in a total of 16 implants placed in 10 participants. Cone-beam computed tomography images show significantly increased bone formation both horizontally and vertically, which provides sufficient support to stabilize implants within 4 months. Together, the findings of our study demonstrate that CelGro is an ideal membrane for GBR not only in oral and maxillofacial reconstructive surgery but also in orthopedic applications (Clinical Trial ID ACTRN12615000027516).

Development of LC chiral methods for neutral pharmaceutical related compounds using reversed phase and normal phase liquid chromatography with different types of polysaccharide stationary phases.

The enantioselectivity of a collection of neutral pharmaceutical compounds on six different types of polysaccharide chiral stationary phases (CSPs), Chiralpak AD (and AD-RH), Chiralcel OD (and OD-RH), Chiralpak OJ (and OJ-R), Chiralcel AS (and AS-RH), Sepapak-2 and Sepapak-4 are compared using reversed phase (RPLC) and normal phase liquid chromatography (NPLC). Screening strategies for maximizing the probability of achieving an initial chiral separation hit for neutral compounds using both RPLC and NPLC are described. Further method optimizations are demonstrated by modifying parameters such as organic modifier composition, eluent pH or CSP particle size. Several practical examples of the application of chiral methods for the study of synthetic reaction mixtures are presented. The most critical validation aspects, including limit of detection, specificity, and ruggedness, are also briefly presented.

Lymphotoxin-beta production following bile duct ligation: possible role for Kupffer cells.

BACKGROUND AND AIMS: Lymphotoxin-beta (LT-beta) may play a role in the pathogenesis of chronic liver injury. The aim of this study was to determine in an animal model of bile duct ligation liver injury whether LT-beta expression is induced and whether Kupffer cells are an intrahepatic source of LT-beta. METHODS: Sprague-Dawley rats were divided into two groups: one group received a single dose of GdCl (a Kupffer cell-blocking agent, 10 mg/kg i.v.), whereas the other group received saline. One day later, the groups underwent bile duct ligation or a sham operation. Liver tissue was obtained on days 1, 3, 5, and 8 for assessment of Kupffer cell numbers, early fibrogenic events and LT-beta gene expression. Kupffer cells were isolated using pronase/collagenase perfusion and centrifugal elutriation. RESULTS: Hepatic LT-beta mRNA expression increased early following bile duct ligation. Pretreatment of bile duct-ligated animals with GdCl significantly reduced the number of Kupffer cells, delayed the rise in LT-beta expression, but had no effect on fibrogenesis. Recovery of the Kupffer cell population in these animals was accompanied by increased hepatic LT-beta expression. The LT-beta ligand and receptor were expressed by isolated normal Kupffer cells. CONCLUSIONS: Hepatic LT-beta expression is induced early following bile duct ligation. Kupffer cells may be an intrahepatic source of LT-beta.

Differential effects of gadolinium chloride on Kupffer cells in vivo and in vitro.

Gadolinium chloride (GdCl) is commonly used to study the role of Kupffer cells in liver disease in vivo. The in vitro effects of GdCl on cultured Kupffer cells are poorly characterised. The aim of this study was to characterise rat Kupffer cell TNFalpha production, phagocytic function, and ED1 and ED2 antigen expression following the administration of GdCl. For in vivo experiments, rats received 10mg/kg GdCl IV or sterile saline. Lipopolysaccharide 3mg/kg IP (LPS) was administered 4h prior to sacrifice on Days 1-3, 5 or 8 following GdCl injection. Hepatic ED1 and ED2 positive macrophage numbers and TNFalpha mRNA levels were determined. For in vitro experiments, Kupffer cells were cultured in the presence of 0-270 microM GdCl for 24h following which viability, TNFalpha protein production in response to LPS (10 ng/ml), phagocytosis, and ED1 and ED2 staining were evaluated. In vivo, the proportion of ED1 positive cells which were ED2 positive was reduced from 87 to 3% and hepatic TNFalpha mRNA levels following LPS declined by 60% over Days 1-5 after injection of GdCl (P<0.01). In vitro, phagocytosis declined with increasing concentrations of GdCl. GdCl (0-27 microM) did not effect cultured Kupffer cell viability, TNFalpha production, ED1 or ED2 staining. We conclude that GdCl significantly reduces ED2 expression by Kupffer cells in vivo. In vitro, GdCl has a dose dependent effect on phagocytosis but only effects viability and TNFalpha production at high concentrations. ED2 expression of cultured Kupffer cells is not affected by GdCl.