Prediction of damage trajectories in systemic sclerosis using group-based trajectory modelling.

OBJECTIVES: Damage accrual in SSc can be tracked using the Scleroderma Clinical Trials Consortium Damage Index (DI). Our goal was to develop a prediction model for damage accrual in SSc patients with early disease. METHODS: Using patients with <2 years disease duration from Canada and Australia as a derivation cohort, and from the Netherlands as a validation cohort, we used group-based trajectory modelling (GBTM) to determine 'good' and 'bad' latent damage trajectories. We developed a prediction model from this analysis and applied it to patients from derivation and validation cohorts. We plotted the actual DI trajectories of the patients predicted to be in 'good' or 'bad' groups. RESULTS: We found that the actual trajectories of damage accumulation for lcSSc and dcSSc were very different, so we studied each subset separately. GBTM found two distinct trajectories in lcSSc and three in dcSSc. We collapsed the two worse trajectories in the dcSSc into one group and developed a prediction model for inclusion in either 'good' or 'bad' trajectories. The performance of models using only baseline DI and sex was excellent with ROC AUC of 0.9313 for lcSSc and 0.9027 for dcSSc. Using this model, we determined whether patients would fall into 'good' or 'bad' trajectory groups and then plotted their actual trajectories which showed clear differences between the predicted 'good' and 'bad' cases in both derivation and validation cohorts. CONCLUSIONS: A simple model using only cutaneous subset, baseline DI and sex can predict damage accumulation in early SSc.

Physical therapy in patients with systemic sclerosis: physical therapists' perspectives on current delivery and educational needs.

OBJECTIVE: To assess the perspectives of physical therapists treating patients with systemic sclerosis (SSc) on their current practice and educational needs. METHOD: In July 2019, 405 SSc patients attending a multidisciplinary SSc programme received a survey on physical therapy. Patients who indicated having received physical therapy in the past 2 years were asked to invite their treating physical therapist to complete a questionnaire including sociodemographic characteristics, referral process, content of treatment, perceived knowledge and skills, and educational needs (mostly yes/no answers). RESULTS: Forty-eight of 80 possibly eligible physical therapists treating SSc patients returned the questionnaire [median age 44 years (interquartile range 35-58); 52% female; median number of SSc patients currently treated: 1 (range 1-4)]. Eighty-one per cent (n = 39) of physical therapists had received a referral, with 69% (n = 27/39) judging its content as insufficient. The most often provided types of exercises were range of motion (96%), muscle-strengthening (85%), and aerobic (71%) exercises, followed by hand (42%) and mouth (10%) exercises. Concerning manual treatment, 65% performed either massage or passive mobilization. Regarding competences, 65% indicated feeling capable of treating SSc patients. Nevertheless, 85% expressed the need for an information website on physical therapy in SSc, and 77% for postgraduate education on SSc. CONCLUSION: Primary care physical therapists treating patients with SSc used a wide range of treatment modalities. Although most stated that they treated very few patients, the majority felt capable of treating SSc patients. Nevertheless, the large majority expressed a need for additional information and educational activities concerning SSc.

Genomic Risk Score impact on susceptibility to systemic sclerosis.

OBJECTIVES: Genomic Risk Scores (GRS) successfully demonstrated the ability of genetics to identify those individuals at high risk for complex traits including immune-mediated inflammatory diseases (IMIDs). We aimed to test the performance of GRS in the prediction of risk for systemic sclerosis (SSc) for the first time. METHODS: Allelic effects were obtained from the largest SSc Genome-Wide Association Study (GWAS) to date (9 095 SSc and 17 584 healthy controls with European ancestry). The best-fitting GRS was identified under the additive model in an independent cohort that comprised 400 patients with SSc and 571 controls. Additionally, GRS for clinical subtypes (limited cutaneous SSc and diffuse cutaneous SSc) and serological subtypes (anti-topoisomerase positive (ATA+) and anti-centromere positive (ACA+)) were generated. We combined the estimated GRS with demographic and immunological parameters in a multivariate generalised linear model. RESULTS: The best-fitting SSc GRS included 33 single nucleotide polymorphisms (SNPs) and discriminated between patients with SSc and controls (area under the receiver operating characteristic (ROC) curve (AUC)=0.673). Moreover, the GRS differentiated between SSc and other IMIDs, such as rheumatoid arthritis and Sjögren's syndrome. Finally, the combination of GRS with age and immune cell counts significantly increased the performance of the model (AUC=0.787). While the SSc GRS was not able to discriminate between ATA+ and ACA+ patients (AUC<0.5), the serological subtype GRS, which was based on the allelic effects observed for the comparison between ACA+ and ATA+ patients, reached an AUC=0.693. CONCLUSIONS: GRS was successfully implemented in SSc. The model discriminated between patients with SSc and controls or other IMIDs, confirming the potential of GRS to support early and differential diagnosis for SSc.

Chemical Modifications of Gold Surfaces with Basic Groups and a Fluorescent Nanoparticle Adhesion Assay To Determine Their Surface p Ka.

For pharmaceutical, biological, and biomedical applications, the functionalization of gold surfaces with pH-sensitive groups has great potential. The aim of this work was to modify gold surfaces with pH-sensitive groups and to determine the p Ka of the modified gold surfaces using a fluorescent nanoparticle adhesion assay. To introduce pH-sensitive groups onto gold surfaces, we modified gold-coated silicon slides with four different bases: 4-mercaptopyridine (4-MP), 4-pyridylethylmercaptan (4-PEM), 4-aminothiophenol (4-ATP), and 2-mercaptoethylamine (2-MEA). To screen whether the modifications were successful, the binding of negatively charged fluorescently labeled nanoparticles to the positively charged surfaces was visualized by fluorescence microscopy and atomic force microscopy. Next, the p Ka of the modified surfaces was determined by quantifying the pH-dependent adhesion of the fluorescently labeled nanoparticles with fluorescence spectroscopy. Fluorescence microscopy showed that the gold surfaces were successfully modified with the four different basic molecules. Moreover, fluorescence spectroscopy revealed that fluorescently labeled negatively charged nanoparticles bound onto gold surfaces that were modified with one of the four bases in a pH-dependent manner. By quantifying the adsorption of negatively charged fluorescently labeled nanoparticles onto the functionalized gold surfaces and using the Henderson-Hasselbalch equation, the p Ka of these surfaces was determined to be 3.7 ± 0.1 (4-MP), 5.0 ± 0.1 (4-PEM), 5.4 ± 0.1 (4-ATP), and 7.4 ± 0.3 (2-MEA). We successfully functionalized gold surfaces with four different basic molecules, yielding modified surfaces with different p Ka values, as determined with a fluorescent nanoparticle adhesion assay.

Interactions of dipalmitoylphosphatidylcholine with ceramide-based mixtures.

The outermost layer of the skin, the stratum corneum (SC), acts as the natural physical barrier. The SC consists of corneocytes embedded in a crystalline lipid matrix consisting of ceramides, free fatty acids and cholesterol. Although phospholipids are frequently present in topical formulations, no detailed information is reported on the interactions between phospholipids and SC lipids. The aim of this study was to examine the interactions between a model phospholipid, dipalmitoylphosphatidylcholine (DPPC) and synthetic ceramide-based mixtures (referred to as SC lipids). (Perdeuterated) DPPC was mixed with SC lipids and the lipid organization and mixing properties were examined. The studies revealed that DPPC participates in the same lattice as SC lipids thereby enhancing a hexagonal packing. Even at a high DPPC level, no phase separated pure DPPC was observed. When a DPPC containing formulation is applied to the skin surface it must partition into the SC lipid matrix prior to any mixing with the SC lipids. To mimic this, DPPC was applied on top of a SC lipid membrane. DPPC applied in a liquid crystalline state was able to mix with the SC lipids and participated in the same lattice as the SC lipids. However, when DPPC was applied in a rippled gel-state very limited partitioning of DPPC into the SC lipid matrix occurred. Thus, when applied to the skin, liquid crystalline DPPC will have very different interactions with SC lipids than DPPC in a (rippled-)gel phase.

Physical activity in patients with systemic sclerosis.

OBJECTIVES: To compare self-reported levels of physical activity (PA) of systemic sclerosis (SSc) patients with the general population. To evaluate in SSc patients factors associated with PA levels and needs and preferences regarding PA. METHODS: Fifty nine SSc patients completed the Short QUestionnaire to ASsess Health-Enhancing PA. The proportion of patients meeting the Dutch Recommendation for PA (= moderate PA for 30 min on ≥ 5 days/week) and total minutes of PA per week were calculated and compared with similar data from the Dutch population. Characteristics were univariately and multivariately compared between patients with low and high PA levels (either ≤ or > mean minutes/week of the Dutch population). Needs and preferences regarding PA promotion and guidance related to exercise were assessed by questionnaires. RESULTS: Stratified for age (< 55 or ≥ 55 years) and gender, the proportion SSc patients meeting the Dutch recommendation for PA was not significantly different from the Dutch population. The total minutes of PA per week was significantly lower among SSc patients (1704 vs. 2614, P < 0.001). Multivariable analyses showed that in SSc patients the male gender, scleroderma health assessment questionnaire (SHAQ) and lack of energy were significantly associated with lower PA levels (P = 0.007; P = 0.042; P = 0.025). Two-third of patients required more information about PA. CONCLUSION: In SSc patients, the total minutes of PA per week are significantly lower compared to the general population. The male gender, functional ability as reflected by SHAQ and lack of energy seem to interfere with PA. These results might guide health professionals in providing their patients with appropriate information on PA.

Stratum corneum lipid matrix: Location of acyl ceramide and cholesterol in the unit cell of the long periodicity phase.

The extracellular lipid matrix in the skin's outermost layer, the stratum corneum, is crucial for the skin barrier. The matrix is composed of ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) and involves two lamellar phases: the short periodicity phase (SPP) and the long periodicity phase (LPP). To understand the skin barrier thoroughly, information about the molecular arrangement in the unit cell of these lamellar phases is paramount. Previously we examined the molecular arrangement in the unit cell of the SPP. Furthermore X-ray and neutron diffraction revealed a trilayer arrangement of lipids within the unit cell of the LPP [D. Groen et al., Biophysical Journal, 97, 2242-2249, 2009]. In the present study, we used neutron diffraction to obtain more details about the location of lipid (sub)classes in the unit cell of the LPP. The diffraction pattern revealed at least 8 diffraction orders of the LPP with a repeating unit of 129.6±0.5Å. To determine the location of lipid sub(classes) in the unit cell, samples were examined with either only protiated lipids or selectively deuterated lipids. The diffraction data obtained by means of D2O/H2O contrast variation together with a gradual replacement of one particular CER, the acyl CER, by its partly deuterated counterpart, were used to construct the scattering length density profiles. The acyl chain of the acyl CER subclass is located at a position of ~21.4±0.2Å from the unit cell centre of the LPP. The position and orientation of CHOL in the LPP unit cell were determined using tail and head-group deuterated forms of the sterol. CHOL is located with its head-group positioned ~26±0.2Å from the unit cell centre. This allows the formation of a hydrogen bond with the ester group of the acyl CER located in close proximity. Based on the positions of the deuterated moieties of the acyl CER, CHOL and the previously determined location of two other lipid subclasses [E.H. Mojumdar et al., Biophysical Journal, 108, 2670-2679, 2015], a molecular model is proposed for the unit cell of the LPP. In this model CHOL is located in the two outer layers of the LPP, while CER EOS is linking the two outer layers with the central lipid layers. Finally the two other lipid subclasses are predominantly located in the central layer of the LPP.

Dissolving Microneedle Patches for Dermal Vaccination.

The dermal route is an attractive route for vaccine delivery due to the easy skin accessibility and a dense network of immune cells in the skin. The development of microneedles is crucial to take advantage of the skin immunization and simultaneously to overcome problems related to vaccination by conventional needles (e.g. pain, needle-stick injuries or needle re-use). This review focuses on dissolving microneedles that after penetration into the skin dissolve releasing the encapsulated antigen. The microneedle patch fabrication techniques and their challenges are discussed as well as the microneedle characterization methods and antigen stability aspects. The immunogenicity of antigens formulated in dissolving microneedles are addressed. Finally, the early clinical development is discussed.

The important role of stratum corneum lipids for the cutaneous barrier function.

The skin protects the body from unwanted influences from the environment as well as excessive water loss. The barrier function of the skin is located in the stratum corneum (SC). The SC consists of corneocytes embedded in a lipid matrix. This lipid matrix is crucial for the lipid skin barrier function. This paper provides an overview of the reported SC lipid composition and organization mainly focusing on healthy and diseased human skin. In addition, an overview is provided on the data describing the relation between lipid modulations and the impaired skin barrier function. Finally, the use of in vitro lipid models for a better understanding of the relation between the lipid composition, lipid organization and skin lipid barrier is discussed. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.

The role of ceramide chain length distribution on the barrier properties of the skin lipid membranes.

The skin barrier function is provided by the stratum corneum (SC). The lipids in the SC are composed of three lipid classes: ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs) which form two crystalline lamellar structures. In the present study, we investigate the effect of CER chain length distribution on the barrier properties of model lipid membranes mimicking the lipid composition and organization of SC. The membranes were prepared with either isolated pig CERs (PCERs) or synthetic CERs. While PCERs have a wide chain length distribution, the synthetic CERs are quite uniform in chain length. The barrier properties were examined by means of permeation studies using hydrocortisone as a model drug. Our studies revealed a reduced barrier in lipid membranes prepared with PCERs compared to synthetic CERs. Additional studies revealed that a wider chain length distribution of PCERs results in an enhanced hexagonal packing and increased conformational disordering of the lipid tails compared to synthetic CERs, while the lamellar phases did not change. This demonstrates that the chain length distribution affects the lipid barrier by reducing the lipid ordering and density within the lipid lamellae. In subsequent studies, the effect of increased levels of FFAs or CERs with a long acyl chain in the PCERs membranes was also studied. These changes in lipid composition enhanced the level of orthorhombic packing, reduced the conformational disordering and increased the barrier of the lipid membranes. In conclusion, the CER chain length distribution is an important key factor for maintaining a proper barrier.

Phase behavior of skin lipid mixtures: the effect of cholesterol on lipid organization.

The lipid matrix in the stratum corneum (SC), the upper layer of the skin, plays a critical role in the skin barrier. The matrix consists of ceramides (CERs), cholesterol (CHOL) and free fatty acids (FFAs). In human SC, these lipids form two coexisting crystalline lamellar phases with periodicities of approximately 6 and 13 nm. In the studies reported here, we investigated the effect of CHOL on lipid organization in each of these lamellar phases separately. For this purpose, we used lipid model mixtures. Our studies revealed that CHOL is imperative for the formation of each of the lamellar phases. At low CHOL levels, the formation of the lamellar phases was dramatically changed: a minimum 0.2 CHOL level in the CER/CHOL/FFA (1 : 0.2 : 1) mixture is required for the formation of each of the lamellar phases. Furthermore, CHOL enhances the formation of the highly dense orthorhombic lateral packing. The gradual increment of CHOL increases the fraction of lipids forming the very dense orthorhombic lateral packing. Therefore, these studies demonstrate that CHOL is an indispensable component of the SC lipid matrix and is of fundamental importance for appropriate dense lipid organization and thus important for the skin barrier function.

Electron diffraction study of lipids in non-lesional stratum corneum of atopic eczema patients.

Skin barrier impairment is thought to be an important factor in the pathogenesis of atopic eczema (AE). The skin barrier is located in the stratum corneum (SC), consisting of corneocytes embedded in lipids. Ceramides, cholesterol and free fatty acids are the major lipid classes and are crucial for the skin barrier function, but their role in relation to AE is indistinct. Filaggrin is an epidermal barrier protein and common mutations in the filaggrin gene strongly predispose for AE. However, there is no strong evidence that filaggrin mutations are related to the reduced skin barrier in AE. In this study, electron diffraction is used in order to study the lipid organization of control SC and non-lesional SC of AE patients in vivo. An increased presence of the hexagonal lipid organization was observed in non-lesional SC of AE patients, indicating a less dense lipid organization. These changes correlate with a reduced skin barrier function as measured with transepidermal water loss but do not correlate with the presence of filaggrin mutations. These results are indicative for the importance of the lipid organization for a proper skin barrier function.

Lipid to protein ratio plays an important role in the skin barrier function in patients with atopic eczema.

BACKGROUND: The barrier function of the skin is primarily provided by the stratum corneum (SC), the outermost layer of the skin. Skin barrier impairment is thought to be a primary factor in the pathogenesis of atopic eczema (AE). Filaggrin is an epidermal barrier protein and common mutations in the filaggrin gene strongly predispose for AE. However, the role of filaggrin mutations in the decreased skin barrier in AE is not fully understood. It was recently shown that changes in SC lipid composition and organization play a role in the reduced skin barrier in AE. OBJECTIVES: To determine whether the lipid/protein ratio and the total dry SC mass per surface area are related to the skin barrier function of controls and patients with AE. METHODS: A case-control study was performed to compare nonlesional and lesional skin of AE with skin of controls. The dry SC mass was determined by tape-stripping and Squamescan(™) . The ratio between lipid and protein bands in the Raman spectrum was used to determine the lipid/protein ratio. Skin barrier function was assessed by transepidermal water loss. RESULTS: The results show that the dry SC mass per skin area is altered only in lesional SC of patients with AE compared with control subjects. The observed reduction in the lipid/protein ratio in SC of patients with AE was more pronounced, both in lesional and nonlesional SC and correlated strongly with the skin barrier function and disease severity. CONCLUSIONS: The lipid/protein ratio plays a role in the reduced skin barrier function in AE.

Intrinsic immunogenicity of liposomes for tuberculosis vaccines: Effect of cationic lipid and cholesterol.

Tuberculosis (TB) is still among the deadliest infectious diseases, hence there is a pressing need for more effective TB vaccines. Cationic liposome subunit vaccines are excellent vaccine candidates offering effective protection with a better safety profile than live vaccines. In this study, we aim to explore intrinsic adjuvant properties of cationic liposomes to maximize immune activation while minimizing aspecific cytotoxicity. To achieve this, we developed a rational strategy to select liposomal formulation compositions and assessed their physicochemical and immunological properties in vitro models using human monocyte-derived dendritic cells (MDDCs). A broad selection of commercially available cationic compounds was tested to prepare liposomes containing Ag85B-ESAT6-Rv2034 (AER) fusion protein antigen. 1,2-Dioleoyl-sn­glycero-3-ethylphosphocholine (EPC)-based liposomes exhibited the most advantageous activation profile in MDDCs as assessed by cell surface activation markers, cellular uptake, antigen-specific T-cell activation, cytokine production, and cellular viability. The addition of cholesterol to 20 mol% improved the performance of the tested formulations compared to those without it; however, when its concentration was doubled there was no further benefit, resulting in reduced cell viability. This study provides new insights into the role of cationic lipids and cholesterol in liposomal subunit vaccines.

Cationic pH-sensitive liposome-based subunit tuberculosis vaccine induces protection in mice challenged with Mycobacterium tuberculosis.

Tuberculosis (TB) has been and still is a global emergency for centuries. Prevention of disease through vaccination would have a major impact on disease prevalence, but the only available current vaccine, BCG, has insufficient impact. In this article, a novel subunit vaccine against TB was developed, using the Ag85B-ESAT6-Rv2034 fusion antigen, two adjuvants - CpG and MPLA, and a cationic pH-sensitive liposome as a delivery system, representing a new TB vaccine delivery strategy not previously reported for TB. In vitro in human dendritic cells (DCs), the adjuvanted formulation induced a significant increase in the production of (innate) cytokines and chemokines compared to the liposome without additional adjuvants. In vivo, the new vaccine administrated subcutaneously significantly reduced Mycobacterium tuberculosis (Mtb) bacterial load in the lungs and spleens of mice, significantly outperforming results from mice vaccinated with the antigen mixed with adjuvants without liposomes. In-depth analysis underpinned the vaccine's effectiveness in terms of its capacity to induce polyfunctional CD4+ and CD8+ T-cell responses, both considered essential for controlling Mtb infection. Also noteworthy was the differential abundance of various CD69+ B-cell subpopulations, which included IL17-A-producing B cells. The vaccine stimulated robust antigen-specific antibody titers, further extending its potential as a novel protective agent against TB.

Localization of cholesterol and fatty acid in a model lipid membrane: a neutron diffraction approach.

The intercellular lipid matrix of the skin's stratum corneum serves to protect the body against desiccation and simultaneously limits the passage of drugs and other xenobiotics into the body. The matrix is made up of ceramides, free fatty acids, and cholesterol, which are organized as two coexisting crystalline lamellar phases. In studies reported here, we sought to use the technique of neutron diffraction, together with the device of isotopic (H/D) substitution, to determine the molecular architecture of the lamellar phase having a repeat distance of 53.9 ± 0.3 Å. Using hydrogenous samples as well as samples incorporating perdeuterated (C24:0) fatty acids and selectively deuterated cholesterol, the diffraction data obtained were used to construct neutron scattering length density profiles. By this means, the locations within the unit cell were determined for the cholesterol and fatty acids. The cholesterol headgroup was found to lie slightly inward from the unit cell boundary and the tail of the molecule located 6.2 ± 0.2 Å from the unit cell center. The fatty acid headgroups were located at the unit cell boundary with their acyl chains straddling the unit cell center. Based on these results, a molecular model is proposed for the arrangement of the lipids within the unit cell.

From the structure of the skin barrier and dermal formulations to in vitro transport models for skin absorption: skin research in the Netherlands and in Germany.

This review presents an overview of German and Dutch research institutions and their studies in the field of skin drug delivery and adjacent topics. In the Netherlands, the involved research groups are mainly localized in Leiden, whereas in Germany the skin research institutions are spread over the whole country. The scientific studies in the Netherlands focus on the in-depth analysis of human skin composition and its individual components as well as on the development and characterization of dermal drug delivery systems ranging from liquid crystalline systems and vesicles up to microneedles with an emphasis on examining the interactions of these drug delivery systems with the human skin in vitro and in vivo. In Germany, the individual areas of research span from in-depth investigations on various drug delivery systems intended for skin application and the development of novel in vitro models for skin absorption testing up to in vivo studies focusing on the biological performance of topically applied actives. Furthermore, sophisticated analytical techniques are applied for the elucidation of skin assembly and transport processes. In addition, experimentally derived data are correlated with advanced computational modelling. Even though the individual research topics in the Netherlands and Germany are quite diverse, the exchange of knowledge and interdisciplinary collaborations between the two neighbouring countries were and are still frequently made. In this context, the review aims at highlighting crosslinks between the different institutions and individual persons to complete the picture. For each institution, the principal investigators and their studies are presented and the upcoming young scientists are introduced as an outlook for the field. This review does not claim completeness, but is rather intended to give a general overview of Dutch and German research in the field of skin drug delivery and adjacent topics.

Rapid radiological progression in the first year of early rheumatoid arthritis is predictive of disability and joint damage progression during 8 years of follow-up.

OBJECTIVE: Several prediction models for rapid radiological progression (RRP) in the first year of rheumatoid arthritis have been designed to aid rheumatologists in their choice of initial treatment. The association was assessed between RRP and disability and joint damage progression in 8 years. METHODS: Patients from the BeSt cohort were used. RRP was defined as an increase of ≥5 points in the Sharp/van der Heijde score (SHS) in year 1. Functional ability over 8 years, measured with the health assessment questionnaire (HAQ), was compared for patients with and without RRP using linear mixed models. Joint damage progression from years 1 to 8 was compared using logistic regression analyses. RESULTS: RRP was observed in 102/465 patients. Over 8 years, patients with RRP had worse functional ability: difference in HAQ score 0.21 (0.14 after adjustment for disease activity score (over time)). RRP was associated with joint damage progression ≥25 points in SHS in years 1-8: OR 4.6. CONCLUSION: RRP in year 1 is a predictor of worse functional ability over 8 years, independent of baseline joint damage and disease activity. Patients with RRP have more joint damage progression in subsequent years. RRP is thus a relevant outcome on which to base the initial treatment decision.

Hyaluronic acid-recombinant gelatin gels as a scaffold for soft tissue regeneration.

An array of different types of hyaluronic acid (HA)- and collagen-based products is available for filling soft-tissue defects. A major drawback of the current soft-tissue fillers is their inability to induce cell infiltration and new tissue formation. Our aim is to develop novel biodegradable injectable gels which induce soft tissue regeneration, initially resulting in integration and finally replacement of the gel with new autologous tissue. Two reference gels of pure HA, monophasic HA-1 and micronised HA-2, were used. Furthermore, both gels were mixed with recombinant gelatin (RG) resulting in HA-1+RG and HA-2+RG. All gels were subcutaneously injected on the back of rats and explanted after 4 weeks. Addition of RG to HA-1 resulted in stroma formation (neovascularisation and ECM deposition) which was restricted to the outer rim of the HA-1+RG gel. In contrast, addition of RG to HA-2 induced stroma formation throughout the gel. The RG component of the gel was degraded by macrophages and giant cells and subsequently replaced by new vascularised tissue. Immunohistochemical staining showed that the extracellular matrix components collagen I and III were deposited throughout the gel. In conclusion, this study shows the proof of principle that addition of RG to HA-2 results in a novel injectable gel capable of inducing soft tissue regeneration. In this gel HA has a scaffold function whereas the RG component induces new tissue formation, resulting in proper vascularisation and integration of the HA-2+RG gel with the autologous tissue.