Study selection and study characteristics

The initial search results included 163 articles: 78 from PubMed and 85 from Scopus. After the removal of 59 duplicates, a total of 104 articles were brought to the screening stage to exclude those that did not meet the eligibility criteria. During the further stage of screening the title and abstract, 52 articles were excluded from the study, since they did not satisfy the inclusion criteria. The remaining 52 articles were subjected to a full-text analysis for the eligibility criteria. As a result of the analysis, 31 articles were found to be ineligible, in particular, 21 of them contained information only on in vitro studies, 2 articles contained only ex vivo experiments, 7 articles did not use a hydrogel matrix, 14 did not use conditioned stem cell medium, and 8 were review articles. Some of the articles contained a combination of the listed ineligibility criteria. Finally, 21 studies were selected for the review. The process of searching and screening the articles is summarized in Fig. 2.

Fig. 2

PRISMA flow diagram representing the selection process of the publications included for the systematic review. Abbreviations used, CM conditioned medium, n number of articles

Further, the articles were categorized for a better understanding of the design and approaches exploited for the fabrication and assessment of regenerating potency of H-CM formulations, involving animal models, and specific wound treatment protocols. Most of the studies represent proof-of-the-concept or concept validation research and describe the hydrogel preparation, CM production and identification of its active components, as well as characterization of the prepared H-CM dressings in vitro and in vivo (Table S1, Supporting Information).

Risk of bias and study quality assessment

According to the result of the CAMARADES quality tool (Table S2, Supporting Information), 19 studies out of 21 (90%) used wound size calculation while assessing the healing efficiency. 8 studies (38%) reported randomization of the experimental and control group allocation. Only 2 included studies (9%) reported the blinded assessment of outcomes. All studies were published in peer-reviewed journals, used appropriate animal models and controls, anesthetized where necessary throughout the study, and stated compliance with the animal welfare regulations. In conclusion, 90% of studies were scored as low risk and 9% were at a medium risk of bias.

According to the Risk of bias (Robvis) tool (Figure S1, Supporting Information), 8 of the 21 studies divided animals into the control and experimental groups randomly and were therefore judged to have a low risk of selection bias. However, none of the articles mentioned that the studies were conducted by assigning, concealing, blinding investigators (unclear risk of bias). Only 2 studies reported blinding of the outcome assessment (low risk of bias). All studies were free from missing data, selective reporting bias, or other biases (low risk of bias). Hence, the quality of the included studies was reliable and acceptable.

Preparing hydrogels loaded with conditioned mediumHydrogel engineering

The natural and synthetic biocompatible and biodegradable polymers are widely used for hydrogel preparation. During the last five years the classical hydrogel-forming components have been gradually replaced by novel synthetic substances and unusual products of natural origin allowing designing various hydrogel-based delivery systems to be used as wound dressings (Fig. 3).

Fig. 3

A five-year retrospective flowchart on the design of wound dressings based on hydrogels loaded with cell-conditioned medium. The panel representing the time point of 2021 is adapted from [52]

In detail, 70% of reviewed studies used mainly natural biopolymers or their chemically modified derivatives such as alginate − 33% [52,53,54,55,56,57,58], chitosan − 19% [59], gelatin − 14% [60], collagen − 14% [61, 62], hyaluronic acid − 5% [63], and/or their combinations [64,65,66]. However, other natural biopolymers such as carrageenan [67], fibrinogen [66], and chondroitin [68] were also found in hydrogel formulations. Rare and unique components of natural origin, e.g., silk fibroin [69], spider silk fusion protein [70], decellularized extracellular matrix (ECM) of porcine skin [71], synthetic polymers like cellulose or its modifications [72], poly(vinyl alcohol) [67], short bioinspired octapeptide [52] or bioceramic materials (e.g., bioglass) [57] were introduced to design hydrogel-based dressings. Within the selection analyzed, the final hydrogels represented mainly soft delivery systems [55, 57, 59, 61, 63,64,65, 70,71,72], or solid bandages [53], sponges [56, 62], membranes [58], or films [54, 66].

The hydrogel structure represents a three-dimensional network which acts as a hydrophilic matrix ensuring prolonged and continuous release of embedded proteins used for tissue regeneration (Table 1). The hydrogel structure is usually homogeneous, but some studies have developed nano-, microstructure-bearing composites, e.g., by using silk fibroin nanofibers [69], or by encapsulating CM components such as extracellular vesicles (exosomes) [56]. Alternatively, multilayer constructs were engineered using the particle-in-particle approach, e.g., alginate microparticles doped with proteins stimulating wound healing, and drug-containing poly(lactic-co-glycolic) acid (PLGA) microspheres to sequentially deliver bioactive molecules [57].

Table 1 Design and structure-functional properties of wound dressings loaded with cell-conditioned medium

Hydrogels containing CM are commonly prepared in their final “ready-to-use” form, however advanced formulations such as in situ-forming grafted hyaluronic acid hydrogels suggest simultaneous crosslinking and gelation directly at the site of application [63]. To prepare a stable hydrogel matrix, their chemical modification or physical treatment is performed. Calcium-based ionic crosslinking in alginate hydrogels [53,54,55, 57, 65] dominates over photopolymerization [60, 63], temperature-induced [64, 69,70,71], freeze-thaw [67], solvent-induced gelation [52] or covalent сrosslinking [62, 68].

Some hydrogels designed were also characterized as microporous materials [52, 53, 60, 68, 70]. The pore diameter was changed by varying the substitution degree and/or concentration of the gel-forming polymer and was shown to affect the release rate of encapsulated proteins of the cell secretome [60, 70]. The mean pore diameter varied greatly from 22 μm to 200 μm. The structure-functional and biopharmaceutical properties such as the protein release kinetics, hydrogel degradation, viscosity and mechanical characteristics of the hydrogels analyzed in the selected articles are shown in Table 1. To enhance the efficiency of the hydrogel treatment, “smart” thermosensitive hydrogels based on chitosan/collagen/β-glycerophosphate hydrogel were also engineered [64]. These matrices were nonfluid at 37 °C and viscous at lower temperatures suggesting a possibility for more effective filling of various types of wounds, including severe burns [64].

Isolation and proteome profiling of cell-derived conditioned medium

In recent decades, numerous studies have demonstrated the beneficial effects of the cell secretome on wound healing [52, 54,55,56, 59, 64, 66,67,68,69, 71, 72], and the number of articles on this topic continues to grow rapidly.

According to the selection analyzed, primary cultures and/or cultures from biobanks or commercially available collections are used for the CM preparation. More than 50% of the selected articles used mesenchymal stem/stromal cells (MSCs) as the secretome sources. Although MSCs are considered to have low immunogenicity [73, 74], recently, there have been a growing number of articles demonstrating that MSCs do not have a full immunological privilege in an immunocompetent allogeneic host [75,76,77]. Therefore, the review also considers other sources of CM including the following animal and human cell types: murine macrophages, in particular RAW 264.7 cells [53, 57], human M2 macrophages derived from monocytes THP-1 [58], dermal fibroblasts [62, 70] and human keratinocytes HaCaT [70], and human embryonic kidney (HEK) 293 cells [65].

The CM production is performed in the lab-scale quantities and based on cell culturing under predetermined conditions using supplemented cell culture media, which may contain additional components to promote cell polarization or growth factors [53, 60, 61]. Prior to the secretome harvesting, an antibiotic component is usually removed from the culture medium. Further, the purification of the obtained medium using centrifugation or filtration is performed to eliminate undesired cell debris. Afterward, the samples are concentrated with a molecular weight cut-off (MWCO) filter. Then, cell CM is prepared for long-term manipulations and storage by freezing at -20 °C – -80 °C or freeze-drying [59, 60, 63, 65, 67, 69, 70]. However, during cell culturing some unusual conditions can be exploited to enrich the medium with cellular factors and bioactive molecules. For example, hypoxic atmosphere [60], gamma-irradiation [78], or transfected cells overexpressing antioxidant proteins (nuclear factor erythroid 2–related factor 2) [65] were used. The typical cell lines, their key characteristics and specific cultivation parameters to prepare cell CM are presented in Table 2. The resultant cell CM product is characterized by a large diversity of its composition, although the proteome profiling and detailed identification of its composition has been performed in several studies [