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Orthopaedic Proceedings
Vol. 102-B, Issue SUPP_6 | Pages 104 - 104
1 Jul 2020
Goodman S Lin T Pajarinen J Yao Z
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Mesenchymal stem cells (MSCs) are capable of forming bone, cartilage and other mesenchymal tissues but are also important modulators of innate and adaptive immune responses. We have capitalized on these important functions to mitigate adverse responses when bone is exposed to pathogen-associated molecular patterns (PAMPs), damage-associated molecular patterns (DAMPs), or prolonged pro-inflammatory cytokines. Our goal was to optimize osteogenesis and mitigate persistent undesired inflammation by: 1. preconditioning MSCs by short term exposure to lipopolysaccharide (LPS) and Tumor Necrosis Factor alpha (TNF-α), 2. genetic modification of MSCs to overexpress Interleukin 4 (IL-4) either constitutively, or as NFκB-responsive IL-4 over-expression cells, and 3. training the MSCs (innate immune memory) by repeated stimulation with LPS.

In the first experiment, bone marrow MSCs and macrophages were isolated from femurs and tibias of C57BL/6 mice. MSCs (1×104 cells) were seeded in 24-well transwell plates in the bottom chamber with MSC growth medium. MSCs were treated with 20 ng/ml TNF-α and 1–20 μg/ml LPS for three days. Primary macrophages (2 × 103 cells) were seeded to the insert of a separate transwell plate and polarized into the M1 phenotype. At day four, MSCs and macrophages were washed and the inserts with M1 macrophages were moved to the plates containing preconditioned MSCs at the bottom of the well. Co-culture was carried out in MSC growth medium for 24h.

In the second experiment, bone marrow derived macrophages and MSCs were isolated from femora and tibiae of Balb/c male mice. 5×104 macrophages and 1×104 MSCs were seeded in the bottom well of the 24-well transwell plate. The upper chambers were seeded with unmodified MSCs, MSCs preconditioned with 20 ng/ml TNF-α and 20 mg/ml LPS for 3 days, NFκB-IL4 secreting MSCs (all 5×104 cells), or controls without MSCs. Co-culture was carried out in mixed osteogenic-macrophage media with clinically relevant polyethylene or titanium alloy particles.

In the third experiment, bone marrow MSCs and macrophages were collected from femurs and tibias of C57BL/6 male mice. The MSCs were stimulated by LPS, washed out for five days, and re-stimulated by LPS in co-culture with macrophages.

First, preconditioned MSCs enhanced anti-inflammatory M2 macrophage (Arginase 1 and CD206) expression, decreased pro-inflammatory M1 macrophage (TNF-α/IL-1Ra ratio) expression, and increased osteogenic markers (alkaline phosphatase expression and matrix mineralization) in co-culture. Second, NFκB-IL4 secreting MSCs decreased pro-inflammatory M1 (TNF-α), increased anti-inflammatory M2 (Arg1, IL-1ra) expression, and enhanced the expression of osteogenic factors Runx2 and alkaline phosphatase, in the presence of particles, compared to other groups.

Third, LPS-trained MSCs increased anti-inflammatory (Arginase1 and CD206), and decreased the proinflammatory (TNF-α, IL1b, iNOS, and IL6) marker expression in MSC/macrophage co-culture.

Transforming MSCs via the techniques of preconditioning, genetic modification, or training (innate immune memory) can modulate/convert a potentially injurious microenvironment to an anti-inflammatory pro-reconstructive milieu. These effects are highly relevant for bone healing in the presence of adverse stimuli. These concepts using transformed MSCs could also be extended to other organ systems subjected to potentially damaging agents.


Bone & Joint Research
Vol. 8, Issue 10 | Pages 481 - 488
1 Oct 2019
Nathan K Lu LY Lin T Pajarinen J Jämsen E Huang J Romero-Lopez M Maruyama M Kohno Y Yao Z Goodman SB

Objectives

Up to 10% of fractures result in undesirable outcomes, for which female sex is a risk factor. Cellular sex differences have been implicated in these different healing processes. Better understanding of the mechanisms underlying bone healing and sex differences in this process is key to improved clinical outcomes. This study utilized a macrophage–mesenchymal stem cell (MSC) coculture system to determine: 1) the precise timing of proinflammatory (M1) to anti-inflammatory (M2) macrophage transition for optimal bone formation; and 2) how such immunomodulation was affected by male versus female cocultures.

Methods

A primary murine macrophage-MSC coculture system was used to demonstrate the optimal transition time from M1 to M2 (polarized from M1 with interleukin (IL)-4) macrophages to maximize matrix mineralization in male and female MSCs. Outcome variables included Alizarin Red staining, alkaline phosphatase (ALP) activity, and osteocalcin protein secretion.


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 42 - 42
1 Jul 2014
Goodman S Yao Z Ren P Gibon E Rao A Pajarinen J Antonios J Lin T Smith R Egashira K Keeney M Yang F Konttinen Y
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Summary

Wear particles from joint replacements may result in loosening and periprosthetic osteolysis. Interference with systemic macrophage trafficking to the implant, modulation of macrophage phenotype from M1 to M2, and inhibition of NFκB may mitigate these adverse effects.

Introduction

Joint replacement of the lower extremity is highly successful in alleviating pain, and improving ambulation and function. However, prosthetic byproducts of different materials, in sufficient amounts, may lead to loosening and periprosthetic osteolysis. Debris from polymers (such as polyethylene and PMMA), metals and ceramics are capable of inciting an adverse tissue reaction, which is orchestrated by cells of the monocyte/macrophage lineage. Three experimental approaches have been taken by our group to potentially mitigate the adverse biological sequela of particle disease. These include: 1) interfering with ongoing migration of monocyte/macrophages to the implant site by inhibiting the chemokine system 2) altering the functional activities of local macrophages by converting pro-inflammatory M1 macrophages to an anti-inflammatory pro-tissue healing M2 phenotype and 3) modulating the production and release of pro-inflammatory cytokines, chemokines and other potentially harmful factors by inhibiting the key transcription factor NFκB.


Orthopaedic Proceedings
Vol. 86-B, Issue SUPP_III | Pages 276 - 276
1 Mar 2004
Savolainen V Pajarinen J Hirvensalo E Lindahl J
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Aims: In the present study we evaluated short-term outcome of complex proximal tibial fractures treated with hybrid external þxation. Methods: A retrospective evaluation of 33 tibiae in 31 patients treated with hybrid external þxation; 9 (27.2%) AO type A metaphyseal and 24 (72.8%) type C bicondylar intraarticular proximal tibial fractures. Majority of the fractures (58.1%) were due to high-energy trauma and open fractures constituted 18.2% of all fractures. Percutaneous pinning combined with indirect reduction techniques was used in 14 (42.4%) cases. Mini-open clamp reduction and screw þxation was applied in 12 (36.4%) operations. Open reduction, reconstruction of articular surface, bone-grafting and screw þxation combined with the hybrid frame was used in 7 (21.2%) patients. Results: Uneventful consolidation without complications occurred in 13 (39.4%) patients. The mean time to bony fusion was 18 ± 6.6 weeks. However, in 21.2% of the patients the fracture had not consolidated by 24 weeks. There were 5 (20.8%) primary re-operations due to malreduction of a type C fracture. In addition 3 (33.3%) type A metaphyseal fractures were re-operated upon due to non-union by 24 weeks. Two (6.1%) fractures united in axial malalignment and required an osteotomy. Conclusions: Our þndings suggest that due to high rate of unsatisfactory reductions, hybrid external þxation may not be the method-of-choice in þxation of displaced intra-articular proximal tibial fractures. It may be indicated in þxation of high energy metaphyseal fractures, but includes a signiþcant risk of delayed consolidation.


Orthopaedic Proceedings
Vol. 86-B, Issue SUPP_III | Pages 293 - 293
1 Mar 2004
Pajarinen J Hirvensalo E
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Aims: To evaluate technical and clinical results of a new 2-incision technique of rotational acetabular osteotomy. Methods: 27 patients, with a mean age of 40 years, were reviewed after a minimum of 1 year subsequently to rotational acetabular osteotomy, which was done through short low midline and iliac approaches. An analysis of complications, the length of rehabilitation period, time to union of the osteotomy, radiographic correction of acetabular roof, proceeding of hip arthrosis, as well as of clinical status at follow-up, using scores by Merle DñAubigne and Harris, was performed. Results: No major operative complications were observed. The median time to union of osteotomy and a painless status was 10 weeks. Femoral head covering by acetabular roof increased signiþcantly, whereas the range of motion of the operated hip was not compromised by the operation. A signiþcant increase in the mean scores, mainly caused by a decrease in pain, were observed in a subgroup of 20 patients with a minimum follow-up of 2 years. The most signiþcant factor predisposing poor outcome was grave preoperative arthrosis of the hip. Conclusions: This new technique of osteotomy is safe and less traumatic than previous methods, allowing early weight bearing and a fast recovery. The early clinical results are satisfactory