Early identification of patients at risk for impaired tendon healing and corresponding novel therapeutic approaches are urgent medical needs. This study aimed to clarify the role of CD3+ T-cells during acute Achilles tendon (AT) healing. Blood and hematoma aspirate were taken from 26 patients during AT reconstruction, and additional blood samples were obtained during clinical follow-up at 6, 26 and 52 weeks after surgery. T-cell subsets were analyzed by flow cytometry using CD3, CD4, CD8, CD11a, CD57 and CD28 antibodies. Clinical follow-up included functional tests, MRI assessments, and subjective questionnaires. In vitro, the functional behavior of patient-derived tenocytes was investigated in co-cultures with autologous unpolarized CD4+ or CD8+ T-cells, or IFNy-polarized CD8+ or IL17-polarized CD4+ Tcells (n=5-6). This included alterations in gene expression (qPCR), MMP secretion (ELISA), migration rate (scratch wound healing assay) or contractility (collagen gels). Analysis revealed that elevated CD4+ T-cell levels and reduced CD8+ T-cell levels (increased CD4/CD8 ratio) in hematoma aspirate and pre-operative blood were associated with inferior clinical outcomes regarding pain and function at 26 and 52 weeks. Increased levels of CD8+ -memory T-cell subpopulations in blood 6 weeks after surgery were associated with less tendon elongation. In vitro, tenocytes showed increased MMP1/2/3 levels and collagen III/I ratio in co-culture with unpolarized and/or IL17-polarized CD4+ T-cells compared to unpolarized CD8+ T-cells. This coincided with increased IL17 receptor expression in tenocytes co-cultured with CD4+ T-cells. Exposure of tenocytes to IL17-polarized CD4+ T-cells decreased their migration rate and increased their matrix contractility, especially compared to IFNy-polarized CD8+ T-cells. The CD4+ /CD8+ T-cell ratio could serve as prognostic marker for early identification of patients with impaired AT healing potential. Local reduction of CD4+ T-cell levels or their IL17 secretion represent a potential therapeutic approach to improve AT healing and to prevent weakening of the tendon ECM.

Bone regeneration is a complex but very well organized process in which the immune system has a decisive role. The adaptive immune system and its experience level (percentage of effector and memory T cells) has been proven to influence the healing cascade especially in the early healing phases. This opens the possibility of an early intervention to enhance bone healing during the primary clinical treatment. Patients stratified for possible delayed bone healing could benefit from immunomodulatory treatment approaches. In pre-clinical studies cells and signaling molecules have been identified that could represent promising candidates to help patients in need.

Mesoporous bioactive glasses (MBGs) have been widely studied as bone regeneration systems, due to their bioactivity and ability to store and release therapeutic agents with specific biological functions. The incorporation of these nanomaterials into a thermosensitive hydrogel (TSH), in which a solution undergoes a sol-gel transition under physiological conditions, represents a promising approach to design multifunctional devices able to deliver selected molecules to pathological sites. In fact, this system can perfectly fit the defect cavity shape prior to the complete gelation, and acts as a carrier for therapeutic agents prolonged release in situ. This challenging concept is the underlying idea of the MOZART project, whose objective was to develop a library of MBGs containing different therapeutic ions and drugs, to be used as a new, smart platform technology for highly targeted therapies to enhance bone healing. The aim of this work is to investigate the bone regeneration potential of MBGs containing strontium ions (pro-osteogenic) and incorporated into thermosensitive poly(etherurethane)(PEU) based on Poloxamer407. In order to further increase the pro-osteogenic response, MBGs were also loaded with N-acetylcysteine (NAC).

MBGs containing 2%mol of Sr²⁺ were prepared by an aerosol-assisted spray-drying method and NAC was loaded post-synthesis via an incipient wetness method. The PEU hydrogel (SHP407) was synthesized via a two-step procedure in nitrogen atmosphere. Particles were characterized (FE-SEM, N₂ adsorption-desorption analysis, TGA, DSC, FT-IR and XRD) and then incorporated into the hydrogel. The hybrid systems rheological properties and stability in aqueous environment at 37°C, and its ability to co-release Sr²⁺ and NAC were analysed. After preliminary biological in vitro tests, a proof-of-concept rodent study was run to assess the ability of the resulting formulation as bone healing device. X-ray at 2 and 4-weeks post-surgery and µCT-analysis were used to evaluate the healing results in a rat osteotomy model of biologically impaired healing. Then, bones were processed for histological evaluation.

Preliminary in vivo results demonstrated that incorporation of MBGs into a TSH is a promising strategy to design a multifunctional injectable formulation for in situ and sustained delivery of pro-osteogenic species enhancing bone regeneration.

Industrialized countries experience a population aging. Elderly patients, due to the experienced immunity, have a constant pro-inflammatory milieu. Little is known on how adaptive immunity impacts the tissue homeostasis and regeneration. The standardized housing of lab animals is specific pathogen free (SPF). However, this housing condition hinders antigen exposure and thus an aging of the adaptive immune system. We hypothesized that exposure to antigens and a developing adaptive immunity will impact tissue homeostasis and regeneration in mice. Mice kept under SPF housing or non-SPF were examined towards their immune status via flow cytometry, bone structure via microCT and bone competence via biomechanical torsional testing. MSCs from these mice were analyzed regarding their differentiation potential and ECM production under various immune cell signaling. Bone regeneration was analyzed in vivo in a mouse osteotomy model. The memory and effector compartment of the adaptive immunity was significantly increased in mice under non-SPF housing. This housing led to an increased femoral cortical thickness and torsional stiffness (p<0,05), whereas the tissue mineral density was not affected. The differentiation potential of stem cells under the influence of an aged immune milieu was significantly reduced. Bone formation was highly affected by the immune status and availed of a naïve immune cell milieu. Adaptive immunity directly impacts bone tissue formation, by exhibiting a constant stress, leading to structural differences in bone tissue organization as well as mechanical competence. For experimental settings, it appears highly relevant if mouse models have had the chance to develop an experienced immune system.

Recently, we could illustrate how tightly the bone and the immune system are interconnected during normal homeostasis but even stronger during bone regeneration. Specifically, the patient´s individual ratio of CD8+ effector T cells (TEFF, already identified as potential unfavorable cells for successful healing) to CD4+ regulatory T cells (TREG, one counterpart to CD8+ TEFF in controlling intratissue inflammation) prior to injury/ surgery appears to determine the healing outcome after fracture. We hypothesized that concentrating CD4+ TREG could serve as innovative therapeutic strategy to improve bone healing. We used an adoptive CD4+ TREG transfer in our well-established mouse osteotomy model. Before treatment, we identified the pre-surgery ratio of CD8+ TEFF/ CD4+ TREG by flow cytometry to characterize the healing potential of individual animals. Thereafter, we performed an adoptive CD4+ TREG transfer to reshape inflammation for supporting osteotomy healing. Across all groups, healing outcome was analyzed after 21 days post-surgery by µCT. Whereas TREG were highly supportive in SPF mice, we observed a heterogeneous clustered healing outcome in the non-SPF mice: TREG responder (improved healing outcome; p = 0.038) and TREG non-responder (impaired healing outcome; p = 0.024). Interestingly, the pre-/peri-surgery ratio of CD8+ TEFF/ CD4+ TREG was higher in the TREG non-responder (p=0.057). Thus, the amount of adoptively transferred CD4+ TREG was not sufficient to improve the healing outcome due to initial unfavorable high CD8+ TEFF/CD4+ TREG ratio. These results clearly show the importance of determining the individual immune status of each patient in the clinic before applying an immunotherapeutic approach.

Despite its intrinsic ability to regenerate form and function after injury, bone tissue can be challenged by a multitude of pathological conditions. While innovative approaches have helped to unravel the cascades of bone healing, this knowledge has so far not improved the clinical outcomes of bone defect treatment. Recent findings have allowed us to gain in-depth knowledge about the physiological conditions and biological principles of bone regeneration. Now it is time to transfer the lessons learned from bone healing to the challenging scenarios in defects and employ innovative technologies to enable biomaterial-based strategies for bone defect healing. This review aims to provide an overview on endogenous cascades of bone material formation and how these are transferred to new perspectives in biomaterial-driven approaches in bone regeneration.

Cite this article: T. Winkler, F. A. Sass, G. N. Duda, K. Schmidt-Bleek. A review of biomaterials in bone defect healing, remaining shortcomings and future opportunities for bone tissue engineering: The unsolved challenge. Bone Joint Res 2018;7:232–243. DOI: 10.1302/2046-3758.73.BJR-2017-0270.R1.

*Winner of ISFR Young Investigator Award

Purpose: The aim of this study was to compare the temporal expression pattern of factors related to cartilage and bone formation and endochondral ossification during standard and delayed bone healing for a more in-depth understanding of the molecular basis of disturbed bone healing and to elucidate suitable timing for substitution of factors to stimulate the healing process.

Methods: A tibial osteotomy was performed in two groups of sheep (n=30 each) and stabilized with either a rigid external fixator leading to standard healing or with a mechanically critical one leading to delayed healing. Hematoma/callus tissue was harvested 4, 7, 14, 21 and 42 days postop. qPCR was employed to determine the expression patterns of BMPs and other molecules.

Results: Gene expressions of BMP2, BMP4, BMP7, Noggin, MMP9 and MMP13 were distinctly lower in the delayed compared to the standard healing group at several time points from day 14, whilst no differential gene expression of Coll II and Coll X was found between both groups. Among the BMPs, BMP7 showed the most markedly differential expression. The first evident difference in BMP7 expression between both groups was found at day 14 suggesting that exogen substitution in the context of a therapeutic approach should be postponed. The differential expression pattern of both MMP9 and MMP13 suggests that there might be a failure or delay in endochondral ossification in delayed bone healing.

Conclusion: Downregulation in gene expression of osteogenic BMPs and cartilage matrix degrading MMPs may account for a considerable delay of bone healing.