Neo-cartilage formation using human nondegenerate versus osteoarthritic chondrocyte-derived cartilage organoids in a viscoelastic hydrogel

Current regenerative cartilage therapies are associated with several drawbacks such as dedifferentiation of chondrocytes during expansion and the formation of fibrocartilage. Optimized chondrocyte expansion and tissue formation could lead to better clinical results of these therapies. In this study, a novel chondrocyte suspension expansion protocol that includes the addition of porcine notochordal cell-derived matrix was used to self-assemble human chondrocytes from osteoarthritic (OA) and nondegenerate (ND) origin into cartilage organoids containing collagen type II and proteoglycans. Proliferation rate and viability were similar for OA and ND chondrocytes and organoids formed had a similar histologic appearance and gene expression profile. Organoids were then encapsulated in viscoelastic alginate hydrogels to form larger tissues. Chondrocytes on the outer bounds of the organoids produced a proteoglycan-rich matrix to bridge the space between organoids. In hydrogels containing ND organoids some collagen type I was observed between the organoids. Surrounding the bulk of organoids in the center of the gels, in both OA and ND gels a continuous tissue containing cells, proteoglycans and collagen type II had been produced. No difference was observed in sulphated glycosaminoglycan and hydroxyproline content between gels containing organoids from OA or ND origin after 28 days. It was concluded that OA chondrocytes, which can be harvested from leftover surgery tissue, perform similar to ND chondrocytes in terms of human cartilage organoid formation and matrix production in alginate gels. This opens possibilities for their potential to serve as a platform for cartilage regeneration but also as an in vitro model to study pathways, pathology, or drug development.</p

Semi-synthetic degradable notochordal cell-derived matrix hydrogel for use in degenerated intervertebral discs: Initial in vitro characterization

Low back pain is the leading cause of disability worldwide, but current therapeutic interventions are palliative or surgical in nature. Loss of notochordal cells (NCs) and degradation of the healthy matrix in the nucleus pulposus (NP), the central tissue of intervertebral discs (IVDs), has been associated with onset of degenerative disc changes. Recently, we established a protocol for decellularization of notochordal cell derived matrix (NCM) and found that it can provide regenerative cues to nucleus pulposus cells of the IVD. Here, we combined the biologically regenerative properties of decellularized NCM with the mechanical tunability of a poly(ethylene glycol) hydrogel to additionally address biomechanics in the degenerate IVD. We further introduced a hydrolysable PEG-diurethane crosslinker for slow degradation of the gels in vivo. The resulting hydrogels were tunable over a broad range of stiffness's (0.2 to 4.5 kPa), matching that of NC-rich and -poor NP tissues, respectively. Gels formed within 30 min, giving ample time for handling, and remained shear-thinning post-polymerization. Gels also slowly released dNCM over 28 days as measured by GAG effusion. Viability of encapsulated bone marrow stromal cells after extrusion through a needle remained high. Although encapsulated NCs stayed viable over two weeks, their metabolic activity decreased, and their phenotype was lost in physiological medium conditions in vitro. Overall, the obtained gels hold promise for application in degenerated IVDs but require further tuning for combined use with NCs

The LRRK2 Arg1628Pro variant is a risk factor for Parkinson's disease in the Chinese population

The c.G4883C variant in the leucine-rich repeat kinase 2 (LRRK2) gene (protein effect: Arg1628Pro) has been recently proposed as a second risk factor for sporadic Parkinson's disease in the Han Chinese population (after the Gly2385Arg variant). In this paper, we analyze the Arg1628Pro variant and the associated haplotype in a large sample of 1,337 Han subjects (834 patients and 543 controls) ascertained from a single referral center in Taiwan. In our sample, the Arg1628Pro allele was more frequent among patients (3.8%) than among controls (1.8%; p = 0.004, OR 2.13, 95% CI 1.29-3.52). Sixty heterozygous and two homozygous carriers of the Arg1628Pro variant were identified among the patients, of which only one was also a carrier of the LRRK2 Gly2385Arg variant. We also show that carriers of the Arg1628Pro variant share a common, extended haplotype, suggesting a founder effect. Parkinson's disease onset age was similar in patients who carried the Arg1628Pro variant and in those who did not carry it. Our data support the contention that the Arg1628Pro variant is a second risk factor for Parkinson's disease in the Han Chinese population. Adding the estimated effects of Arg1628Pro (population attributable risk [PAR] ∼4%) and Gly2385Arg variants (PAR ∼6%) yields a total PAR of ∼10%

Notochordal cell matrix as a therapeutic agent for intervertebral disc regeneration

Notochordal cells (NCs) reside in the core of the healthy disc and produce soluble factors that can stimulate nucleus pulposus cells (NPCs). These NC-derived factors may be applied in intervertebral disc regeneration for treatment of low-back pain. However, identification of the active soluble factors is challenging. Therefore a novel approach to directly use porcine NC-rich NP matrix (NCM) is introduced. We explored porcine NCM's anabolic effects on bovine NPCs harvested from caudal discs of adolescent and adult (2-2.5 vs. 4-6 year old) cows. NC-conditioned medium (NCCM) and NCM were produced from porcine NC-rich NP tissue. Bovine NPCs were cultured in alginate beads for 4 weeks in base medium (BM), NCCM, and NCM to investigate NCM's regenerative potential. Porcine NCM increased glycosaminoglycan (GAG) content of both adolescent and adult bovine NPCs. This was through increased proliferation of adolescent bovine NPCs, whereas in adult bovine NPCs, it was mostly through increased GAG production per NPC. Furthermore, adolescent bovine NPCs were cultured in BM and porcine NCM treated with interleukin (IL)-1β to investigate NCM's potential in an inflammatory environment. Addition of IL-1β enhanced IL1β and CXCL8 (IL8) gene expression, while NCM diminished IL1β gene expression. IL-1β reduced GAG and DNA content, but the addition of NCM relative to BM improved GAG and DNA content. Altogether, porcine NCM exerts bovine NPC-age dependent effects, and NCM's anabolic effect on adult NPCs is stronger compared with NCCM. Furthermore, porcine NCM induced an anabolic response of bovine NPCs in an inflammatory environment and may have anti-inflammatory properties. Therefore, NCM has potential in a regenerative therapy for disc degeneration, and warrants additional in vivo studies

Notochordal Cell Matrix As a Therapeutic Agent for Intervertebral Disc Regeneration

Notochordal cells (NCs) reside in the core of the healthy disc and produce soluble factors that can stimulate nucleus pulposus cells (NPCs). These NC-derived factors may be applied in intervertebral disc regeneration for treatment of low-back pain. However, identification of the active soluble factors is challenging. Therefore a novel approach to directly use porcine NC-rich NP matrix (NCM) is introduced. We explored porcine NCM's anabolic effects on bovine NPCs harvested from caudal discs of adolescent and adult (2–2.5 vs. 4–6 year old) cows. NC-conditioned medium (NCCM) and NCM were produced from porcine NC-rich NP tissue. Bovine NPCs were cultured in alginate beads for 4 weeks in base medium (BM), NCCM, and NCM to investigate NCM's regenerative potential. Porcine NCM increased glycosaminoglycan (GAG) content of both adolescent and adult bovine NPCs. This was through increased proliferation of adolescent bovine NPCs, whereas in adult bovine NPCs, it was mostly through increased GAG production per NPC. Furthermore, adolescent bovine NPCs were cultured in BM and porcine NCM treated with interleukin (IL)-1β to investigate NCM's potential in an inflammatory environment. Addition of IL-1β enhanced IL1β and CXCL8 (IL8) gene expression, while NCM diminished IL1β gene expression. IL-1β reduced GAG and DNA content, but the addition of NCM relative to BM improved GAG and DNA content. Altogether, porcine NCM exerts bovine NPC-age dependent effects, and NCM's anabolic effect on adult NPCs is stronger compared with NCCM. Furthermore, porcine NCM induced an anabolic response of bovine NPCs in an inflammatory environment and may have anti-inflammatory properties. Therefore, NCM has potential in a regenerative therapy for disc degeneration, and warrants additional in vivo studies

The Stimulatory Effect of Notochordal-Cell Conditioned Medium in a Nucleus Pulposus Explant Culture

OBJECTIVES: Notochordal cell-conditioned medium (NCCM) has previously shown to have a stimulatory effect on nucleus pulposus cells (NPCs) and bone marrow stromal cells (BMSCs) in alginate and pellet cultures. These culture methods provide a different environment than the nucleus pulposus (NP) tissue, in which the NCCM ultimately should exert its effect. The objective of this study is to test whether NCCM stimulates NPCs within their natural environment, and whether combined stimulation with NCCM and addition of BMSCs has a synergistic effect. METHODS: Bovine NP tissue was cultured in an artificial annulus in base medium (BM), porcine NCCM, or BM supplemented with 1 µg/ml Link N. Furthermore, BM and NCCM samples were injected with 1 million BMSCs per NP sample. Samples were cultured for 4 weeks, and analyzed for biochemical contents (water, GAG, hydroxyproline, and DNA), gene expression (COL1A1, COL2A1, ACAN, and SOX9), and histology by Safranin O/Fast Green staining. RESULTS: Culture in NCCM resulted in increased proteoglycan content compared to day 0 and BM, similar to Link N. However, only minor differences in gene expression compared to day 0 were observed. Addition of BMSCs did not result in increased GAG content, and surprisingly, DNA content in BMSC injected groups was not higher than in the other groups after 4 weeks of culture. DISCUSSION: This study shows that indeed, NCCM is capable of stimulating NPC matrix production within the NP environment. The lack of increased DNA content in the BMSC-injected groups indicates that BMSCs have died over time. Identification of the bioactive factors in NCCM is crucial for further development of a NCCM-based treatment for intervertebral disc regeneration

Notochordal cell matrix: An inhibitor of neurite and blood vessel growth?

Blood vessel and neurite ingrowth into the degenerating intervertebral disc (IVD) are related to pain. In reported studies, notochordal cell (NC)-conditioned medium (NCCM) induced a regenerative response of nucleus pulposus (NP) cells, but also inhibition of neurite and vessel formation. NC matrix (NCM) derived from NC-rich NP tissue, induced even stronger anabolic effects than NCCM. Thus, the aim was to investigate whether NCM has similar anti-neurogenic and -angiogenic properties as NCCM. NCM and NCCM where produced from porcine NC-rich NP tissue. Human umbilical vein endothelial cells (HUVECs) were cultured in base medium (BM, 300 mOsm), NCCM (produced at 300 and 400 mOsm), NCM, or with chondroitin sulfate (CS, positive control) in angiogenesis-inducing medium, after which vessel length was measured. Although CS alone inhibited vessel growth, NCCM (both osmolarities) stimulated vessel formation by HUVECs. NCM did not affect vessel growth relative to BM. SH-SY5Y cells were cultured in BM, NCCM, and NCM on poly-D-lysine coated and polystyrene surfaces, and analyzed for neurite length and percentage of neurite expressing cells. On coated surfaces, neither NCCM nor NCM affected neurite growth. On a polystyrene surface, NCCM and NCM induced a higher number of neurite-expressing cells. NCCM's previously reported anti-angiogenic and -neurogenic effects were not observed in this study. Although addition of CS inhibited HUVEC vessel formation, other factors may be present in NCCM and NCM that affect neurite and vessel growth. Therefore, future studies testing an NC-based regenerative strategy should carefully assess the risk of such adverse effects in an in vivo setting

The Stimulatory Effect of Notochordal Cell-Conditioned Medium in a Nucleus Pulposus Explant Culture

Objectives: Notochordal cell-conditioned medium (NCCM) has previously shown to have a stimulatory effect on nucleus pulposus cells (NPCs) and bone marrow stromal cells (BMSCs) in alginate and pellet cultures. These culture methods provide a different environment than the nucleus pulposus (NP) tissue, in which the NCCM ultimately should exert its effect. The objective of this study is to test whether NCCM stimulates NPCs within their native environment, and whether combined stimulation with NCCM and addition of BMSCs has a synergistic effect on extracellular matrix production. Methods: Bovine NP tissue was cultured in an artificial annulus in base medium (BM), porcine NCCM, or BM supplemented with 1 μg/mL Link N. Furthermore, BM and NCCM samples were injected with 106 BMSCs per NP sample. Samples were cultured for 4 weeks, and analyzed for biochemical contents (water, glycosaminoglycan [GAG], hydroxyproline, and DNA), gene expression (COL1A1, COL2A1, ACAN, and SOX9), and histology by Safranin O/Fast Green staining. Results: Culture in NCCM resulted in increased proteoglycan content compared to day 0 and BM, similar to Link N. However, only minor differences in gene expression compared to day 0 were observed. Addition of BMSCs did not result in increased GAG content, and surprisingly, DNA content in BMSC-injected groups was not higher than in the other groups after 4 weeks of culture. Discussion: This study shows that, indeed, NCCM is capable of stimulating NPC matrix production within the NP environment. The lack of increased DNA content in the BMSC-injected groups indicates that BMSCs have died over time. Identification of the bioactive factors in NCCM is crucial for further development of an NCCM-based treatment for intervertebral disc regeneration

Identifying potential patient-specific predictors for anterior cruciate ligament reconstruction outcome – a diagnostic in vitro tissue remodeling platform

Purpose Upon anterior cruciate ligament (ACL) rupture, reconstruction is often required, with the hamstring tendon autograft as most widely used treatment. Post-operative autograft remodeling enhances graft rupture risk, which occurs in up to 10% of the patient population, increasing up to 30% of patients aged under 20 years. Therefore, this research aimed to identify potential biological predictors for graft rupture, derived from patient-specific tissue remodeling-related cell properties in an in vitro micro-tissue platform. Methods Hamstring tendon-derived cells were obtained from remnant autograft tissue after ACL reconstructions (36 patients, aged 12–55 years), and seeded in collagen I gels on a micro-tissue platform. Micro-tissue compaction over time – induced by altering the boundary constraints – was monitored. Pro-collagen I expression was assessed using ELISA, and protein expression of tenomodulin and α-smooth muscle actin were measured using Western blot. Expression and activity of matrix metalloproteinase 2 were determined using gelatin zymography. Results Only micro-tissues corresponding to younger patients occasionally released themselves from the constraining posts. Pro-collagen I expression was significantly higher in younger patients. Differences in α-smooth muscle actin and tenomodulin expression between patients were found, but these were age-independent. Active matrix metalloproteinase 2 expression was slightly more abundant in younger patients. Conclusions The presented micro-tissue platform exposed patient-specific remodeling-related differences between tendon-derived cells, with the micro-tissues that released from constraining posts and pro-collagen I expression best reflecting the clinical age-dependency of graft rupture. These properties can be the starting point in the quest for potential predictors for identifying individual patients at risk for graft rupture