**3. Model for a traffic monitoring system**

This section introduces an analytical model which works out some characteristics of network traffic analysis systems. There are several alternatives to model theoretically this type of system. For example, you can use models of queuing theory, Petri nets and, even, mixed models. The ultimate goal is to have a theoretical model that allows us to study the performance of a network traffic analysis system, considering those parameters that are the most representative: throughput, number of processors, analysis load and so on.

We have chosen a theoretical model based on closed queuing networks. It is able to represent accurately the behaviour of a system in charge of analysing network traffic loaded in a multiprocessor architecture. Queuing theory allows us to develop models in order to study the performance of computer's systems [Kobayashi, 1978]. Proposed model consists in a closed queue network where CPU consumptions are related to the service capacity of the queues.

It is worth mentioning that both the flowing traffic and the processing capacity at the nodes are modelled by Poisson arrival rates and exponential service rates. Poisson's distributions are considered to be acceptable for modelling incoming traffic [Barakat et al., 2002]. This assumption can be relaxed to more general processes such as MAPs (Markov Arrival Processes) [Altman et al., 2000], or non homogeneous Poisson processes, but we will keep working with it for simplicity of the analysis. Regarding service rate modelling, although program's code has a quite deterministic behaviour, some randomness is introduced by Poisson incoming traffic, variable length of packets and kernel scheduler uncertainty.


**1. Introduction**

Hydrogels are a group of biomaterials that are perceived as a valuable tool in both cosmetology and dermatology, as they are efficient carriers of various therapeutic substances used in the treatment of a wide range of skin diseases [1,2]. This is mainly due to their ease of use and minimal range of possible side effects that are often observed in oral or intravenous drug administration. The multitude of hydrogel matrices currently under development enables many active substances, both hydrophilic and hydrophobic, to be incorporated into their structures [3]. This can be achieved through the formation of inclusion complexes, the production of nanoparticles, liposomes, microspheres and/or micelles [1,3–6]. Hydrogels based on natural and synthetic polymers, owing to their high biocompatibility, non-toxicity, biofunctionality, biodegradability, relatively low immunogenicity as well physical properties similar to natural tissues, are used for materials with many biomedical applications [1], namely, for the production of contact lenses, artificial organs and materials for the reconstruction and regeneration of cartilage, for tissue engineering and reconstructive surgery, as dressings for the healing of wounds as well as

**Citation:** Zagórska-Dziok, M.; Kleczkowska, P.; Ol˛edzka, E.; Figat, R.; Sobczak, M. Poly(chitosan-esterether-urethane) Hydrogels as Highly Controlled Genistein Release Systems. *Int. J. Mol. Sci.* **2021**, *22*, 3339. https://doi.org/10.3390/ijms22073339

Academic Editor: Geoffrey Brown

Received: 25 February 2021 Accepted: 22 March 2021 Published: 24 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

release systems for various compounds with therapeutic effects [1,7–13]. In recent years, hydrogel biomaterials that are characterized by static properties or play the role of "smart" hydrogels that can respond to different types of stimuli have been more and more dynamically developed [14]. Furthermore, there is also an increasing interest in this type of material in the treatment of various dermatological diseases and beauty deficiencies. Indeed, the use of hydrogels as carriers of medicinal agents, both local and systemic, may prove to be an effective tool against skin diseases, which has been indicated by numerous scientific reports [15–17].

A number of forms of hydrogels containing numerous pharmacologically active substances in their structures and intended for use in the treatment of numerous skin diseases have been developed. These biomaterials also have a positive effect on the wound healing process and are used as fillers in aesthetic medicine treatments and as scaffolds for tissue regeneration [18–26]. Genistein (GEN) is an isoflavonoid with a wide spectrum of biological activity, which was first isolated from the *Genista tinctoria* L. in 1899, but is also present in many raw materials of plant origin [27]. Being a phytoestrogen makes it interesting as a potent valuable material for cosmetology as well as dermatology [28]. Estrogen receptors are found in various areas of the skin, where they play a specific, welldefined role. The similarity of the chemical structure of GEN to estrogens allows it to bind to estrogen receptors located in the cell nucleus, which results in a change in the expression of many genes that play an important role in many physiological and metabolic processes of the body [29]. Based on the current scientific reports, it can be concluded that the incorporation of GEN into the structure of hydrogels may prove helpful in the context of dermatology and cosmetology, because this phytoestrogen may contribute to the inhibition of skin aging processes [30,31], reduce the appearance of brown "age spots" on the body and improve the elasticity and firmness of our skin [32,33]. Additionally, the possibility of inhibiting the multiplication of many microorganisms by GEN indicates that this compound may also prove to be a useful tool in the fight against various bacterial and fungal diseases of the skin [34–37].

Many research groups have also attempted to evaluate the effects of GEN by conducting both in vitro and in vivo analysis. The obtained results indicate that in vitro GEN can inhibit the excessive growth of fibroblasts, which prevents their excessive proliferation and the formation of unsightly scars [38]. Research also indicates that GEN may counteract the inhibition of collagen biosynthesis by fibroblasts and protect skin cells from the harmful effects of UV radiation [39,40]. In addition, this compound reduces inflammation, improves the wound healing process, reduces oxidative and photodynamic damage and promotes DNA repair processes [32,41,42]. Research on skin ageing has also shown that GEN can inhibit this process by increasing the thickness of collagen fibers, increasing the resistance of the skin to breakage and reducing the levels of TGF-b1, MMP-2, MMP-9, VEGF, TIMP-1 and TIMP-2 [43,44]. Furthermore, the clinical trials showed that isoflavones, including GEN, have a positive effect on the proliferation of epidermal cells, the production of collagen, the reduction of fine wrinkles and the improvement of skin elasticity [41].

Even though GEN has skin protecting activity, its sustained effect is still limited due to lower stability in cosmeceutical products [45]. In order to enhance stability and prolong activity, delivery technologies have greater impact in cosmeceutical sectors. For example, GEN-loaded nanoemulsions were prepared with 250 nm sized nanoparticles and enhanced delivery of isoflavones to the skin with higher skin protecting activity [46]. In another study, nanosized liposome-encapsulated GEN in nanoparticles the size of about 80 nm was tested on rat skin, with or without hair [47]. In addition to the nanosized liposomes, the hairs on the skin also affect the delivery of the GEN compound to the skin [45]. No commercial hydrogels containing genistein dedicated to cosmetology and dermatology have been yet developed. This is probably due to the unsatisfactory control of the kinetics of genistein release from such carriers. However, there is still intensive work on this type of materials in many scientific and industrial centers.

Given the above, in this work, we have shown for the first time new synthesized poly(chitosan-ester-ether-urethane) hydrogels characterized by highly GEN controlled release. A significant novelty of our findings relies on using biodegradable hydrogels with different structures as an efficient solution for the modification of GEN-release properties. It is important to mention that the obtained hydrogels possess a desired safety profile in terms of lack of cytotoxicity towards skin cells. We believe that such obtained hydrogels could be practically applied for dermatology and cosmetology.

#### **2. Results and Discussion**

#### *2.1. Synthesis of CL, LA and PEG Copolymers*

The main aim of the present work was the synthesis and characterization of poly(chitosanester-ether-urethane) hydrogels as new and potential GEN carriers. Our main intention was to establish a relationship between the composition and physicochemical properties of the synthesized hydrogels and the amount of GEN released from them. As a consequence, the hydrogels were obtained by a three-step method.

In the first step (1), copolymers *rac*-lactide (*rac*-LA) and poly(ethylene glycol) (PEG) (PLA-PEG) or ε-caprolactone (CL) and PEG (PCL-PEG) were synthesized via enzyme Ring-Opening Polymerization process (e-ROP) (Table 1). The e-ROP process was carried out at 80 ◦C during 7 days in toluene as a medium. Immobilized lipase B from *Candida antarctica* (CALB) was used as a biocatalyst. The molar ratio of the monomers (*rac*-LA or CL) to PEG was: 20:1, 25:1, 30:1, 35:1 or 40:1. The chemical structure of the obtained copolymers was confirmed by 13C, 1H NMR and FTIR studies (Experimental section). As shown in Table 1, the synthesized copolymers were characterized by a similar value of Mn (3200–3700 g/mol), but a different content of PEG units in the polymer chain (circa 11, 15 and 20 mol. % PEG). Reaction yields ranged from 69 to 88%.

**Table 1.** Synthesis of CL, *rac*-LA and PEG copolymers.

a *rac*-LA (or CL): PEG 400 molar ratio; b average molar mass and dispersity determined by the GPC method; c percent of mers molar content in the chain (determined by 1H NMR);Reaction conditions: temp. 80 ◦C, time—7days, medium—toluene, CALB (400 mg).

#### *2.2. Swelling and Biodegradation of Hydrogels Studies*

In the next stage of this study, the hydrogels were synthesized using prepolymer methods such as: (2) preparation of prepolymers and (3) polyaddition of prepolymers and chitosan (CHT) (Scheme 1). In step (2), various PLA-PEG or PCL-PEG were reacted with hexamethylene diisocyanate (HDI) in a −NCO/−OH molar ratio of 2.05:1. In step (3), the prepolymer was reacted with CHT in a −NCO(prepolymer)/−OH(or NH2) (CHT) ratio of 1.5:1. The dibutyltin dilaurate (DBDLSn) was used as a polyaddition catalyst (Table 2).

The swelling capacity of the obtained hydrogels was determined (Table 2). The value of the coefficient of the mass swelling ratio (MSR) was 375%, 311%, 284%, 297%, 258% and 211% after 4 h for HPUCHT-1, HPUCHT-2, HPUCHT-3, HPUCHT-4, HPUCHT-5 and HPUCHT-6, respectively. As we can easily see, the value of the MSR depended on the nature of the polyester used and the content of the PEG units in the hydrogel chain. As expected, MSR increases with the growth of hydrophilic fragments—PEG units in the polymer chain.

**Scheme 1.** Hydrogel synthesis.

**Table 2.** Characterization of hydrogels.

Reaction conditions: temperature—80 ◦C, time—3 h (the first step of the prepolymer process) and 4 h (the second step the of prepolymer process); HDI and PLA-PEG 400 (or PCL-PEG 400) molar ratio: 2.05:1 (in a −NCO/−OH); −NCO(prepolymer)/−OH(or −NH2) (CHT) molar ratio: 1.5:1; MSR—mass swelling ratio. a—after 4 h; b—after 24 h; c—after 48 h.

#### *2.3. In Vitro Release Studies of GEN from Hydrogels*

GEN was loaded into hydrogels using the incorporation method. The mean weight of the devices developed was approximately 200 mg, corresponding to approximately 10 mg of GEN (active substance content was adjusted to about 5%). In vitro studies of the release of GEN from obtained hydrogel materials were determined at pH 7.4 and 37 ◦C for 80 h (Figure 1). The ordinate of the plot was calculated based on the cumulative amount of GEN released with respect to its initial amount in the hydrogels.

**Figure 1.** GEN release profiles from the hydrogels.

As mentioned above, our intention in this work was to investigate the influence of two factors on the kinetic release of GEN. The first one was the content of the PEG units in the copolymer; the second was the nature and properties of the polymer forming the copolymer (PLA or PCL).

The rate of the GEN release decreased as follows: HPUCHT-1 (obtained from PLA-PEG-1 (80:20)) > HPUCHT-2 (PLA-PEG-2 (85:15)) > HPUCHT-3 (PLA-PEG-3 (88:12)) > HPUCHT-4 (PCL-PEG-1 (80:20)) > HPUCHT-5 (PCL-PEG-2 (84:16)) > HPUCHT-6 (PCL-PEG-3 (89:11)).

The data points obtained for the drug release studies were subject to zero-order and first-order kinetics as well as to the Korsmeyer−Peppas models for the evaluation of the kinetic and mechanism of GEN release from hydrogels (Table 3). As is known from the literature, according to the Korsmeyer−Peppas model, for the diffusion-degradationcontrolled drug release system, the release exponent value n is in the range of 0.45 and 0.89 (anomalous, non-Fickian), whereas when n is close to 0.45, the diffusion (Fickian diffusion) predominates in the process and, in the opposite case, n > 0.89, the model corresponds to the super case II transport [48,49].

**Table 3.** Analysis data of GEN release from the obtained hydrogels.

In our study, it was observed that the kinetic release of GEN is mainly influenced by the content of the PEG units in the copolymers used for the preparation of hydrogels. For example, approximately 80.8%, 73.4% and 65.2% of GEN were released after 24 h from the HPUCHT-1, HPUCHT-2 and HPUCHT-3 samples, respectively. This is relatively little, because 60.7%, 58.3% and 56.8% of GEN were released after 24 h from the HPUCHT-4, HPUCHT-5 and HPUCHT-6 materials composed of PCL segments in different molar ratios (Table 2). Thereby, we can conclude that GEN was released more rapidly from materials created of PLA polymers than from PCL segments. After 49, 53 and 55 h, almost 100% of

GEN was released from hydrogels composed of PLA units (Figure 1). In the case of PCL polymer materials, the time for the GEN releasing in 100% was longer and ranged up to 78 h (Figure 1). This is undoubtedly related to the higher hydrophilicity of hydrogels obtained from PLA-PEG copolymers compared with those obtained from PCL-PEG copolymers. The Mn of the PLA-PEG or PCL-PEG copolymers used in the hydrogel synthesis did not appear to affect the amount of GEN released. This could be because the Mn values of all copolymers were similar (3200–3700 g/mol) (which was consistent with the authors' assumptions). It was also discovered that the higher the PEG content in a given matrix composed of PLA or PCL units, the higher the amount of GEN released.

The GEN release kinetic from hydrogels obtained from PCL-PEG copolymers (HPUCHT-4, HPUCHT-5, HPUCHT-6) followed the near-zero-order model (R2 was 0.923, 0.925 and 0.941, respectively). Furthermore, for the HPUCHT-3 sample (hydrogel containing the lowest percentage of PEG units, but composed of PLA units), the GEN release was also close to the zero-order kinetics (R2 = 0.921). Furthermore, it was noted that GEN was released from the HPUCHT-1 and HPUCHT-2 samples with rather first-order kinetics (R2 was 0.915 and 0.940 (Table 3). The analysis of GEN release data using the Korsmeyer−Peppas model suggested that all the hydrogels were governed rather by a non-Fickian transport (n = 0.452–0.609).

The biodegradation of the blank hydrogels has also been carried out. Hydrolytic degradation test of the resulting carriers was conducted under the same conditions as the GEN release experiments. The degradation process was characterized by the plotting of the weight loss (WL) of hydrogels versus time. The results are shown in Figure 2.

**Figure 2.** The weight loss (WL) of the obtained hydrogels.

The degradation rate of the hydrogels was found to be similar to that of the GEN release profile (Figure 1). The blank hydrogels degradation rate was as follows: HPUCHT-1 > HPUCHT-2 > HPUCHT-3 > HPUCHT-4 > HPUCHT-5 > HPUCHT-6. Similarly, hydrogels formed of PLA polymer degrade faster than that obtained from PCL segments. Apparently, this corresponds to a higher hydrophilicity of the PLA polymer. In addition, the rate of degradation of the hydrogel has increased with an increase in the content of the PEG units in the copolymer chain. In summary, on the basis of the data obtained from the kinetic release and mathematical models, it can be concluded that in some cases, GEN was released with relatively high control from the synthesized hydrogel materials.

Certain GEN vectors, including nanoparticles, nanocapsules or nanoemulsions, have been developed and thoroughly studied [50–54]. For instance, GEN was released from zein and zein/carboxymethyl CHIT nanoparticles at a rate of about 60% after 24 h. The authors suggest a controlled release, although the "burst release" phenomenon can be observed in the kinetic profiles. Moreover, the authors did not match the obtained release results with any kinetic models [50]. GEN-loaded solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) were obtained by Andrade et al. [50]. Skin permeation studies have shown that lipid nanoparticles have increased GEN skin retention but GEN release kinetics have not been fully controlled [50]. GEN-loaded PLA nanocapsules

(GEN-NC) were prepared in paper [52] by interfacial deposition of pre-formed polymers (nanoprecipitation). Permeation experiments have demonstrated that a higher amount of GEN reaches deeper layers of the skin and increased penetration was achieved when GEN-NC was incorporated into a semi-solid gel formulation. It indicated that GEN-NC could be a promising nanocarrier system for GEN skin delivery, but this active substance release kinetics was not fully controlled. Nanoemulsions composed of isopropyl myristate/dioleylphosphaditylcholine/oleylamine with GEN have also been obtained [53]. The incorporation of GEN into nanoemulsions significantly increased the retention of this isoflavone in epidermis and dermis. These results were supported by confocal images. Such formulations exhibited antiherpetic activity in vitro against herpes simplex virus 1 and herpes simplex virus 2. The results shows that the GEN-loaded nanoemulsions developed in this study are promising options for herpes treatment. Nanoemulsions containing isoflavone aglycone-rich fraction (NE IAF) and derivative semi-solid hydrogels composed of hyaluronic acid have been obtained at work [54]. NE IAF containing GEN has been prepared and, in some formulations, hyaluronic acid has been added to obtain hydrogels. The distribution of GEN in skin layers has been evaluated. These results showed the potential of formulations for topical skin applications. However, the kinetics of GEN release from the above-mentioned carriers have not been investigated.

In the light of the cited literature examples, we are unfortunately of the opinion that the comparison of these biomaterials with the hydrogels produced in our work is quite difficult due to the fact that our materials have been synthesized from a different type of polymer and thus have a different structure. In our view, the developed biodegradable poly(chitosanester-ether-urethane) hydrogels (HPUCHT-3, HPUCHT-4, HPUCHT-5, HPUCHT-6) are characterized by relatively high release control and may constitute a potential material for further dermatological applications.

### *2.4. Genotoxic Test*

The umu-test was used to evaluate the genotoxicity of the blank hydrogels (Table 4). In the umu-test, it was found that none of the samples tested were toxic to *S. typhimurium* (G > 0.5) (Table 4). None of the tested extracts exhibited a genotoxic effect (IR < 1.5) with or without metabolic activation (IR < 1.5).

**Table 4.** The results of the umu-test for the extracts tested.

\* without metabolic activation; \*\* with metabolic activation.

### *2.5. Cytotoxicity Assessment*

#### 2.5.1. Neutral Red Uptake Assay

In order to assess the biocompatibility of the obtained hydrogels and the possibility of their use in the treatment of various skin diseases, cytotoxicity tests were carried out on skin cells located in different layers of the skin—fibroblasts and keratinocytes. For this purpose, these cells were exposed to the tested hydrogels for 2 and 24 h. The cell viability was estimated using the Neutral Red (NR) and Alamar Blue (AB) assays. The obtained results revealed that the hydrogels tested were characterized by high biocompatibility,

as no cytotoxic effects were observed in any of the hydrogels. The analysis conducted showed that the effect of the experiments on keratinocytes (HaCaT) and fibroblasts (BJ) varies depending on the type of hydrogel tested and the time of exposure to hydrogel extracts. The NR assay performed on fibroblasts showed that the extended exposure time to the hydrogels tested had a positive effect on the viability of these cells, which is probably related to the release of GEN from hydrogels and its positive effect on the skin cells of interest. Notably, promising results for HPUCHT-3 and HPUCHT-4 hydrogels were observed after a shorter exposure time. An average cell viability of 188% was recorded for HPUCHT-3 hydrogel at the highest extract concentration (5%). After longer exposure, all the hydrogels tested were found to stimulate an increase in the viability of the fibroblasts. However, in the case of HPUCHT-6 hydrogel, this effect was observed only at the two highest extract concentrations (2.5 and 5.0%) (Figure 3A,B). In the case of keratinocytes, such a significant effect of the prolonged exposure time on the increase in the viability of these cells was unnoted (Figure 4A,B). Increased viability of keratinocytes treated with hydrogels tested was also reported to be lower compared to fibroblasts. The most promising results have, indeed, been achieved for HPUCHT-2, HPUCHT-3 and HPUCHT-4 hydrogels. On the other hand, the remaining hydrogels did not have a positive effect on the viability of HaCaT cells as a response to the extended exposure time, but still had no cytotoxic activity (Figure 4B).

#### 2.5.2. Alamar Blue Assay

In the case of the Alamar Blue assay, no cytotoxicity of the analyzed hydrogels was observed for eithertypes of skin cells. These analyses showed that the longer exposure time (up to 24 h) to the tested hydrogels significantly increased the metabolic activity of the fibroblasts, which reached 165 and 175% for HPUCHT-3 and HPUCHT-4 hydrogels at the highest concentrations (Figure 5A,B). On the other hand, better results were obtained for a shorter incubation time for keratinocytes, indicating a higher sensitivity of these cells to the action of the hydrogels tested.

**Figure 3.** Effect of different concentrations of extracts (0.1–5%) from hydrogels on NR uptake in cultured fibroblasts after 2 h (**A**) and 24 h (**B**) of exposure. \*\*\*\* *p* ≤ 0.0001, \*\*\* *p* ≤ 0.001, \*\* *p* ≤ 0.01, \* *p* ≤ 0.05 versus the control (100%).

**Figure 4.** Effectof different concentrations of extracts (0.1–5%) from hydrogels on NR uptake in cultured HaCaT cells after 2 h (**A**) and 24 h (**B**) of exposure. \*\*\*\* *p* ≤ 0.0001, \*\*\* *p* ≤ 0.001, \*\* *p* ≤ 0.01, \* *p* ≤ 0.05 versus the control (100%).

**Figure 5.** Thereduction of resazurin after 2 h (**A**) and 24 h (**B**) of exposure to extracts (0.1–5%) from hydrogels in cultured fibroblasts. \*\*\*\* *p* ≤ 0.0001, \*\*\* *p* ≤ 0.001, \*\* *p* ≤ 0.01, \* *p* ≤ 0.05 versus the control (100%).

It should be noted, however, that no cytotoxic effect was observed after 2 and 24 h of exposure of keratinocytes to the hydrogels tested and most of the hydrogels stimulated the metabolic activity and viability of these cells (Figure 6A,B). The studies carried out, thus, show a lack of in vitro cytotoxicity of the hydrogels tested, which gives hope for the possibility of their potential application after appropriate in vivo analyses and clinical trials.

**Figure 6.** Thereduction of resazurin after 2 h (**A**) and 24 h (**B**) of exposure to extracts (0.1–5%) from hydrogels in cultured HaCaT cells. \*\*\*\* *p* ≤ 0.0001, \*\*\* *p* ≤ 0.001, \*\* *p* ≤ 0.01, \* *p* ≤ 0.05 versus the control (100%).

As part of the studies, the effect of GEN on the cell lines tested was also assessed. The obtained results indicate that this compound has no negative effect on both keratinocytes and fibroblasts in the tested concentration range (1–1000 µM). In the case of fibroblasts, it was observed that a 2-h incubation of these cells with GEN causes a statistically significant increase in the metabolic activity of these cells (the concentrations was ranged from 100 to 750 µM), and after 24-h incubation with 100 µM of GEN, the increase in this activity was almost 70% compared to the control cells (untreated with GEN). Concentrations of 250–1000 µM also significantly increased the proliferation of fibroblasts. However, as the concentration of GEN increased, this effect was weaker, which could indicate a cytotoxic effect of GEN at higher concentrations (above 1000 µM) (Figure 7A,B). In the case of keratinocytes, no statistically significant increase in cell proliferation was observed after 2-h incubation, while the longer incubation period resulted in an increase in the metabolic activity of these cells exposed to GEN at concentrations of 1–500 µM. Higher concentrations (750 and 1000 µM) did not cause a decrease in cell proliferation compared to control cells, but, as in the case of fibroblasts, a downward trend in the activity of these cells was observed along with an increase in the GEN concentration (Figure 8A,B).

**Figure 7.** The reduction of resazurin after 2 h (**A**) and 24 h (**B**) of exposure to GEN (1–1000 µM) in cultured fibroblasts. \*\*\*\* *p* ≤ 0.0001, \*\*\* *p* ≤ 0.001 versus the control (100%).

**Figure 8.** The reduction of resazurin after 2 h (**A**) and 24 h (**B**) of exposure to GEN (1–1000 µM) in cultured HaCaT cells. \*\*\*\* *p* ≤ 0.0001, \*\*\* *p* ≤0.001,\* *p* ≤ 0.05 versus the control (100%).

The increase in cell viability and the rate of proliferation following the use of GEN may be due to an improvement in the mitochondrial membrane potential and reduced release of reactive oxygen species by increasing the glutathione (GSH) level, as indicated by other authors [33]. The use of hydrogel matrices directly to the skin may also prove to be an effective route of administration of this isoflavone, as GEN has low bioavailability and is rapidly metabolized following oral administration, which has a significant effect on the absorption of this compound [55,56]. This is supported by the results obtained by Huang et al., which demonstrate that topical administration may be an effective route for the delivery of soy isoflavones, including GEN [57]. Consequently, the possibility of controlled release of GEN from the biodegradable hydrogels developed in this study, with the simultaneous lack of their cytotoxic effect on skin cells, may suggest the possibility of their potential use in the treatment of dermatological diseases after more detailed in vivo and clinical studies. Taking into account the noticeable lack of effective systems for controlled release of GEN, which has an extremely beneficial effect on the skin described in the introduction to this work, the hydrogels developed may be an opportunity to improve the skin condition of many people currently struggling with common skin diseases.

#### **3. Materials and Methods**

#### *3.1. Materials*

1,6-diisocyanatohexane (hexamethylene diisocyanate, HDI, 98%, Aldrich, Poznan, Poland), 3,6 dimethyl 1,4 dioxane 2,5 dione (*rac*-lactide, *rac*-LA, 99%, Sigma-Aldrich, Poznan, Poland), acetic acid (CH3COOH, ≥99%, Sigma-Aldrich, Poznan, Poland), antibiotics (Penicillin-Streptomycin, Life Technologies, Bleiswijk, The Netherlands), ε Caprolactone, (2 Oxepanone, CL, 99%, Aldrich, Poznan, Poland), chitosan (CHT, low molecular weight, 75% deacetylated), dibutyltin dilaurate (DBDLSn, >96%, Sigma-Aldrich, Poznan, Poland), dichloromethane (DCM, CH2Cl2, ≥99.8%, POCh, Gliwice, Poland), DMEM (Dulbecco's Modification of Eagle's Medium, Biological Industries, Beit Haemek, Israel), ethyl alcohol (ethanol, C2H5OH, 96%, Sigma-Aldrich, Poznan, Poland), FBS (Fetal Bovine Serum, Biological Industries, Genos, Lodz, Poland), genistein (GEN, >98%, Tokyo Chemical Industry Co. LTD., Tokyo, Japan), hydrochloric acid (HCl, ChemPur, Piekary Sl ˛askie, Poland), immobi- ´ lized lipase B from *Candida antarctica* (CALB) (Sigma-Aldrich, Poznan, Poland), Neutral Red Solution (NR, 0.33%, Sigma-Aldrich, Poznan, Poland), *N*,*N*-dimethylformamide (DMF, anhydrous, 99.8%, Sigma-Aldrich, Poznan, Poland), phosphate buffered saline (PBS, pH 7.00 ± 0.05, ChemPur, Piekary Slaskie, Poland), poly(ethylene glycol) 400 (PEG 400, Sigma- ´ Aldrich, Poznan, Poland), resazurin sodium salt (RES, Sigma-Aldrich, Poznan, Poland), toluene (99.8%, POCh, Gliwice, Poland) and trypsin-EDTA solution (Sigma-Aldrich, Poznan, Poland) were used as received.

#### *3.2. Synthesis of CL, LA and PEG Copolymers*

The polymerization reactions were carried out according to our previously described method with some modifications [58–60]. Before the reaction, monomers (CL or rac-LA), PEG and CALB were dried under vacuum at room temperature for 3 h. Next, 0.04 mol CL (or 0.04 mol *rac*-LA) was placed in a three-neck flask equipped with a stirrer and thermometer (under argon atmosphere) and 20 mL of toluene was added. The mixture was stirred at 60 ◦C for 2 h. Next, an appropriate amount of PEG 400 and CALB (400 mg) was added to the mixture (Table 1). Stirring was continued at 60 ◦C for 24 h under argon atmosphere. After this time, the enzyme was filtered off. Toluene was removed by evaporation under reduced pressure at room temperature. Next, the cooled product was dissolved in DCM and extracted within cold methanol and distilled water.

#### *3.3. Spectroscopy Data*

The 1H NMR spectrum of *rac*-LA and PEG copolymer (PLA-PEG): 1.57 ppm (-O(O)C-CH(CH3)-), 3.63 ppm (O CH2-CH2-O-), 4.29 ppm (-O(O)C-CH(CH3)-OH end group) and 5.17 ppm (-O(O)C-CH(CH3)-).

The 13C NMR spectrum of PLA-PEG: 16.7 ppm (-O(O)C-CH(CH3)-), 69.0 ppm (-O(O)C-CH(CH3)-), 70.5 ppm (-O-CH2-CH2-O) and 169.3 ppm (-O(O)C-CH(CH3)-).

The FTIR spectrum of PLA-PEG (KBr, cm−1 ): 2997 (υasCH3), 2947 (υsCH3), 2882 (υCH), 1760 (υC=O), 1452 (δasCH3), 1348–1388 (δsCH3), 1368-1360 (δ1CH+δsCH3), 1315- 1300 (δ2CH), 1270 (δCH + υCOC), 1215-1185 (υasCOC + rasCH3), 1130 (rasCH3), 1100-1090 (υsCOC), 1045 (υC-CH3), 960-950 (rCH3 + υCC), 875-860 (υC-COO), 760-740 (δC=0), 715- 695 (γC=O), 515 (δ1C-CH3 + δCCO), 415 (δCCO), 350 (δ2C-CH3 + δCOC), 300-295 (δCOC + δ2C-CH3), 240 (τCC).

The 1H NMR spectrum of CL and PEG copolymer (PCL-PEG): 1.38 ppm (O CH2-CH2- CH2-CH2-CH2-),1.65 ppm (-O-CH2-CH2-CH2-CH2-CH2-), 2.31 ppm (-O(O)C-CH2-CH2- CH2-CH2-CH2-), 3.65 ppm (-O-CH2-CH2-O-) from PEG mers and (CH2 CH2-CH2-CH2- CH2-OH end groups), 4.06 ppm (-O-CH2-CH2-CH2-CH2-CH2-) and 4.23 ppm (-O-CH2- CH2-O(O)C-CH2-CH2-CH2-CH2-CH2-).

The 13C NMR spectrum of PCL-PEG: 24.7 ppm (-O(O)C-CH2-CH2-CH2-CH2-CH2-), 25.6 ppm (-O(O)C-CH2-CH2-CH2-CH2-CH2) 28.4 ppm (-O-CH2-CH2-CH2-CH2-CH2-), 34.2 ppm(-O(O)C-CH2-CH2-CH2-CH2-CH2-), 64.2 ppm (-O-CH2-CH2-CH2-CH2-CH2-), 70.6 ppm(-O-CH2-CH2-O-) and 173.6 ppm (-O(O)C-CH2-CH2-CH2-CH2-CH2-).

The FTIR spectrum of PCL-PEG (KBr, cm−1 ): 2944 (νasCH2), 1722 (νC=O), 1244 (νC-O).

#### *3.4. Hydrogels' Preparation*

The hydrogels were obtained according to the described prepolymer method with some modifications [61]. The prepolymers were obtained through a polyaddition reaction between HDI and PLA-PEG (or PCL-PEG) in a −NCO/−OH molar ratio of 2.05:1, using 2 drops of 0.1 wt% DBDLSn solution in toluene as catalyst. The reactions were performed at 80 ◦C for 3 h under argon atmosphere to form an isocyanate terminated prepolymer. Next, the dispersion of CHT into glacial acetic acid/DMF mixture (30 mL) in a volume ratio of 50/50 has been prepared. Next, the obtained prepolymer was added to the dispersion of CHT. The reactions were carried out in a −NCO(prepolymer)/−OH (or NH2) (CHT) molar ratio of 1.5:1 at 80 ◦C for 4 h under argon atmosphere. The reaction mixture was then transferred to the distilled water. Precipitated products were separated by filtration and washed with DMF, methanol and acetone. The final products were dried under vacuum for one week. The developed hydrogels were denoted as: HPUCHT-1 (formed from PLA-PEG-1, 80:20), HPUCHT-2 (formed from PLA-PEG-2, 85:15), HPUCHT-3 (formed from PLA-PEG-3, 88:12), HPUCHT-4 (formed from PCL-PEG-1, 80:20), HPUCHT-5 (formed from PCL-PEG-2, 84:16) and HPUCHT-3 (formed from PCL-PEG-3, 89:11).

#### *3.5. Swelling and Biodegradation of Hydrogels Studies*

The mass swelling ratio (MSR) of hydrogels was determined at 37 ◦C during 80 h of incubation in PBS. Samples (approximately 0.5 g) in triplicate were submerged in a PBS solution (10 mL) for a given time, and their weights were taken after removing the excessive surface water. The mass swelling ratio was calculated using the following formula:

$$\text{MSR} = ((\text{W2} - \text{W1}) \, / \, \text{W1}) \times 100\%$$

where:

W1 is the weight of the initial hydrogel;

W2 is the weight of the swollen hydrogel.

In order to evaluate the percentage of degradation, the hydrogel samples were immersed in PBS at 37 ◦C for 4 weeks; most importantly, the medium was replaced with fresh PBS every one week. At the end of the experiment, the samples were dried in a vacuum for 48 h. The degree of degradation of hydrogels (in triplicate) was determined by the weight loss (WL) of the samples according to the equation:

$$\text{WL} = [(\text{W1} - \text{W2}) \, / \, \text{W1}] \times 100\%$$

where:

WL is the weight loss;

W1 is the weight of dry sample before degradation; W2 is the weight of the dry sample after degradation.

#### *3.6. In Vitro Release Studies of GEN from Hydrogels*

GEN was loaded to the hydrogel matrices by physical mixing due to the following procedure. A total of 5.0 % (m/m) of GEN in distilled water/Tween 80 mixture (2% (*w*/*v*) was added to six hydrogel samples (HPUCHT-1, HPUCHT-2, HPUCHT-3, HPUCHT-4, HPUCHT-5 and HPUCHT-6). The hydrogels were left sealed for 24 h. The mixtures were dried under vacuum at room temperature to obtain a GEN-loaded hydrogel films. The in vitro release of GEN from the hydrogels was performed in a PBS buffer (pH 7.4 ± 0.05) containing 2% (*w*/*v*) Tween 80 at 37 ◦C under stirring. Vials containing hydrogel films were filled with 5.0 mL of PBS buffer (pH 7.4 ± 0.05), sealed and left in 37 ◦C for 2 h. The solutions were then removed for further testing and replaced by fresh PBS. Subsequent samples were collected at selected intervals. GEN concentration in the in vitro samples was also determined by HPLC (detected at the wavelength of 262 nm). The mobile phase was composed of 70% methanol and 30% water with 0.1% phosphoric acid [62,63]. The release data points were subjected to zero-order, first-order kinetics and Korsmeyer–Peppas models, respectively. Calculations were made on the basis of formulas mentioned below:

Zero-order model:

F=kt

First-order model:

$$
\log \text{F} = \log \text{F}\_0 - \text{kt/2.303}
$$

Korsmeyer–Peppas model:

$$\text{F=kt}^{\text{n}} \text{ (F<0.6)}$$

where:

F is the fraction of GEN released up to time (t);

F0 is the initial concentration of GEN;

k is the constant of the mathematical models;

n is the exponent of the Korsmeyer–Peppasmodel [48,49].

#### *3.7. Measurements*

The structures of synthesized materials were characterized using 1H and 13C NMR techniques (Varian 300 MHz, Palo Alto, CA, USA). The spectra were registered in CDCl3. The 1H spectra were recorded under following conditions: 300 MHz, 1 s repetition time, 2 s acquisition time, 32, 64 or 128 scans. The 13C spectra: 75.4 MHz, 5 s repetition time, 1.4 s acquisition time, 20,000 scans per each spectrum. Percentage molar value of PEG in a copolymer chain were estimated using the following formulas [64]:

$$\% \, CL = \frac{\frac{I\_{\rm{PEG}}}{2}}{I\_{\varepsilon} + \frac{I\_{\rm{PEG}}}{2}} \cdot 100\%$$

$$\% \, LA = \frac{\frac{I\_{\rm{PEG}}}{4}}{I\_{\alpha} + \frac{I\_{\rm{PEG}}}{4}} \cdot 100$$

The FTIR spectra of PLA-PEG and PCL-PEG prepolymers were measured from KBr pellets (PerkinElmer spectrometer, Great Britain). The average molecular weight and molecular weight distribution were determined on the LabAllianceGel Permeation Chromatograph equipped with Jordi Gel DVB mixed bed (250 × 10 mm) column and refractive detector, using chloroform as solvent. The flow rate was 1 mL/min. The average molecular weights were calibrated with polystyrene standards.

#### *3.8. Genotoxic Test*

The umu-test is a bioassay to evaluate the genotoxic potential of environmental samples and chemical compounds. The test detects the induction of the SOS system to the strain *Salmonella typhimurium* TA1535/pSK1002. The SOS system is the bacterial response to the DNA-damaging agents. The test strain is genetically modified- the *umuC* gene activity is linked to the synthesis of β-galactosidase, while other DNA regions responsible for the synthesis of this enzyme were deleted. Therefore, β-galactosidase activity strictly depends on the SOS system induction level and the genotoxic activity of the tested sample [65]. Additionally, the bacteria growth (G) is evaluated by a measurement of an optical density to determine the cytotoxicity of tested samples. The genotoxic potential of the sample was presented as the Induction Ratio (IR)-the β-galactosidase activity of the tested sample relative to the negative control. Samples with IR ≥ 1.5 are considered as genotoxic.

In the present study, the umu-test was carried out in the micro-plate variant according to the ISO guideline, with and without metabolic activation (S9 liver fraction) [66]. Deionized sterile water was used as a negative control, 2-aminoanthracene and 4-nitroquinoline N-oxide were used as positive controls, and phosphate buffered saline (PBS from Gibco, Thermo Fisher Scientific, Darmstadt, Germany) as solvent control. All tested samples were incubated in PBS—100 mg/mL for 24 h, at 37 ◦C, with shaking. Before the assay, all extracts were sterilized by filtration (0.20 µm). All samples were tested in two-fold dilution series (four concentrations in three replicates; the highest concentration of 66.6 mg/mL).

#### *3.9. Cell Culture and Preparation of Hydrogel Extracts*

Two human skin cell lines were used in the study: HaCaT cells (normal human keratinocytes; CLS Cell Lines Service, Eppelheim, Germany) and BJ cells (fibroblasts, ATCC®CRL-2522™; the American Type Culture Collection, Manassas, VA, USA). Both cell lines were maintained in DMEM medium supplemented with L-glutamine, 4.5 g/L glucose and sodium pyruvate. In order to achieve optimal results, the culture medium was supplemented with 10% (*v*/*v*) FBS and 1% (*v*/*v*) antibiotics (100 U/mL penicillin and 1000 µg/mL streptomycin). Cells were cultured in an incubator at 37 ◦C in a humidified atmosphere of 95% air and 5% carbon dioxide (CO2). After the cultured cells (HaCaT and BJ) had reached proper confluence (about 70–80%), the DMEM culture medium was removed from the culture plate (VWR) and the cells were washed twice with sterile PBS. Subsequently, the cell layer was trypsinized with trypsin/EDTA, and the cells were then resuspended in fresh DMEM medium. In the next step, cells were plated in 96-well flatbottom plates (separate plates for both cell types) and left for 24 h in the incubator. After HaCaT and fibroblasts were attached to the bottom of the plates, cells were incubated with various concentrations of extracts (0.1, 0.5, 1.0, 2.5 and 5.0%) obtained from six different hydrogels (HPUCHT-1, HPUCHT-2, HPUCHT-3, HPUCHT-4, HPUCHT-5, HPUCHT-6) loaded with GEN. Extracts were obtained by incubating test hydrogels in DMEM culture medium for 24 h on a rocker shaker, after whichappropriate dilutions of these extracts were prepared in DMEM. Before the assay started, all extracts were sterilized by filtration (0.20 µm). The cells were then treated with the hydrogel extracts for 2 and 24 h in an incubator. Control samples were cells grown in the DMEM medium without the addition of hydrogel extracts.

#### *3.10. Cytotoxicity Assays*

#### 3.10.1. Neutral Red Uptake Assay

In order to evaluate the cytotoxicity of the tested hydrogels on HaCaT and BJ cells, the Neutral Red Uptake Assay (Sigma Aldrich, Poznan, Poland) was used. This test was performed based on the procedure described previously [67]. After 2 and 24 h of exposure to extracts of the six GEN-loaded hydrogels, cells were incubated for 2 h with a neutral red dye (40 µg/mL), which was dissolved in serum-free DMEM medium. After incubation with NR, cells were washed with sterile PBS and 150 µL of decolorizing buffer (C2H5OH/CH3COOH/H2O, 50%/1%/49%) was added to each well to release cellular dye

into the PBS solution. After shaking the cells for 15 min the absorbance of the dissolved dye at λ = 540 nm was determined using a FilterMax F5 Multi-Mode microplate reader (Thermo Fisher). The mean absorbance of the control cells (untreated with the extracts) was taken as 100% cell viability and used to calculate the percentage of viable cells in the experimental samples treated with the test hydrogels. As a part of the study, three independent experiments were carried out, in which each concentration of extracts was tested using four replicates.

#### 3.10.2. Alamar Blue Assay

The cytotoxicity assessment of the hydrogels tested on skin cells was also carried out using the Alamar Blue test. The protocol described by Page et al. [68] has been used for this purpose. After 2 and 24 h of exposure of HaCaT and BJ cells (on separate 96-well plates) to individual concentrations of the analyzed hydrogel extracts (0.1 to 5% concentration range), a resazurin solution (with a final concentration of 60 µM) was added to the well and incubated for 2 h at 37 ◦C in the dark. The fluorescence of the samples was then measured at λ = 570 nm using a microplate reader (FilterMax F5, Thermo Fisher). The experiments were performed in three independent experiments, in which the fluorescence of the cells in four wells was measured for each extract concentration. The results were expressed as a percentage of cell viability compared to the control sample (100%), which were cells grown in DMEM medium without any addition of hydrogel extracts. Additionally, in order to assess the effect of GEN (not loaded into hydrogel structures) on the tested cells, fibroblasts and keratinocytes were incubated with GEN in the concentration range of 1–1000 µM for 2 and 24 h and the cytotoxicity assessment was performed.

#### *3.11. Statistical Analysis*

Values of different parameters were expressed as the mean ± standard deviation (SD). Two-way analysis of variance (ANOVA) and Bonferroni posttest between groups were performed at the level *p* < 0.05 to evaluate the significant differences between values. Statistical analyses were performed using GraphPad Prism 8.4.3 (GraphPad Software, Inc., San Diego, CA, USA).

#### **4. Conclusions**

In the present work, new non-toxic and biodegradable hydrogels for controlled release of GEN have been obtained and characterized. The developed materials have been obtained using a three-step method. The poly(ester-ether)s components have been synthesized by an e-ROP process catalyzed by CALB. In the next steps, hydrogels were obtained by a prepolymer method using CHT and HDI, in the presence of DBDLSn as a catalyst. The in vitro release study showed that the release rate of GEN was highly dependent on the composition of newly developed poly(chitosan-ester-ether-urethane) hydrogels. We also found that, in some cases, GEN was released with relatively high control, near-zero-order kinetics. The "burst release" of GEN has not been observed. Importantly, in vitro studies conducted on the skin cells have shown that the hydrogels do not show a cytotoxic effect on both fibroblasts and keratinocytes. In addition, the GEN-loaded hydrogels have a positive effect on the viability and proliferation of these cells. From a broader perspective, this study suggests that synthesized hydrogels may potentially be used in dermatology and cosmetology.

**Author Contributions:** Conceptualization: M.Z.-D. and M.S.; methodology, M.Z.-D., P.K., E.O., R.F. and M.S.; validation, M.Z.-D., P.K., E.O. and M.S.; formal analysis, M.Z.-D., P.K., E.O. and M.S.; investigation, M.Z.-D., P.K., E.O., R.F. and M.S.; data curation, M.Z.-D., R.F. and M.S.; writing original draft preparation, M.Z.-D. and M.S.; writing—review and editing, M.Z.-D., P.K., E.O. and M.S.; visualization, M.Z.-D. and M.S.; supervision, M.S.; project administration, M.Z.-D. and M.S.; funding acquisition, M.Z.-D. and M.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was founded by the 503-07-01-34 Statutory Project of the University of Information Technology and Management in Rzeszow, Poland.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


Rubrics can be used several times throughout a project and at least once midway through. Students should self-evaluate their projects or potentially evaluate the projects of peers. If you do peer evaluations, set expectations that critical feedback should be accompanied by positive feedback. Review feedback before sharing it to screen potentially hurtful comments. After applying the rubric, provide time for students to reflect on the rubric outcomes. Reflection questions can include:

Revisiting team contracts and doing team check-ins (Chapter 6) effectively provide formative assessment focused on psychosocial axes.

At the end of a project, students can go through the rubric and reflection process again, using both as evidence to propose a grade (if needed). Some of this process can parallel, or occur at the same time as, overall project reflection (Chapter 11). Reflection questions can include:

Students can make their grade proposals via a written document or via a conversation with you. By allowing revision of work without penalty,

248

or by reducing the potential impact of a single grade on the student's overall success, this process can focus on growth-centered evaluation. You can adjust a student's grade from their proposal if needed, but doing this frequently indicates you should improve your rubric/ reflection process.

In inquiry projects, providing revision opportunities can be difficult because of time or place-access constraints. Do a formative assessment early so that students have maximum time available to revise their projects.

# *Grade-Level Differentiation*

#### **Elementary (3–5)**

Students can share "wows" (what they liked/were impressed by) and "wonders" (what else they imagine would be interesting) about their peers' projects.

# *Examples*

*Figure 13.3* Providing peer feedback on projects

*Figure 13.4* Written peer feedback on an observation

*Figure 13.5* Post-project journaling and reflection

*Figure 13.6* Reflecting on a project at sunset

# **14 Conclusion**

# **14.1 What Students and Teachers Say**

Place-based scientific inquiry is an effective and accessible tool for teaching and learning outside. It can make a real difference for your students. Here are a few viewpoints from partners, primarily at Title I–eligible schools in Arizona:

You know, this is the way science should be taught to us. The real-world experience has made science more exciting to me instead of the boring stuff we read in books. I could really get into being a scientist if I got to do this type of work every day.

– Student

[I developed] a sense of wonder. . . we literally had things to wonder about that we could sit in [the forest's] shade and rip the leaves off them kinda thing, that's not the same thing with having the models.. . . Studying models is not as cool as studying trees.

– Student

[T]he data analysis. . . was really cool because we're actually using it to, like, answer questions.. . . [But with classroom] homework, it was just answering random questions …

– Student

What helped me a lot was the hands-on component. In school, most of the kids are fully sighted, right? Most things are watching or seeing only, where we were out in the field doing experiments and testing things and feeling things, which helped a lot.

– Student (visually impaired)

[Doing inquiry] has been an amazing experience for me. . . [it] has already made me less anxious about leaving home for college and made me understand I can study whatever I want.

– Student

I think having the students collect data to answer a question. . . is an important thing for young scientists to know how to do. The data collection. . . engaged students. I also appreciated that the students were also given time to explore and ask questions of their own. [This can] .. . offer my students so much—the opportunity to be outdoors, do field science, engage in scientific inquiry. . .

– Teacher

I love the way [this helps]. . . the kids to make observations, formulate hypotheses, and then figure out a way to collect data and evidence to support conclusions.. . . I love the way you all used the [outdoors]. . . to teach science concepts.. . . It is the perfect setting to encourage student exploration, curiosity, and discovery.

– Teacher

[These] methods are new to me, and I enjoyed seeing the students develop community, knowledge of social justice, environmental issues, and diversity. – Teacher

[This is] a great opportunity to start and create real and lasting changes in our communities.

– Teacher

# **14.2 Overcoming Common Fears**

*If you do not know what students could study*, take a walk around the school or the neighborhood, and try one of the observation/question generation activities. Every place can be interesting and valuable with open eyes.

*If you do not know how much time you will need*, start with a small and structured project, and follow the sample schedules. Budget at least a month in advance to think through your planning. Double your initial time estimates for active instruction to accommodate the unforeseen.

*If you are worried about risk and discomfort outside,* start close by. Pick places that are close to indoor spaces (e.g., school playgrounds) and take frequent breaks.

*If you feel that you do not know enough about the natural world, scientific equipment, or the focal place*, you can learn alongside your students. Start with simpler projects and simpler tools and familiar places. You can model the process of learning alongside your students.

*If you think that you cannot manage many student groups simultaneously*, start with a whole-class inquiry project where multiple small groups work together on the same question. Or invest time in securing volunteers so you can reach a viable student-to-adult ratio.

*If you think that your students will lose respect for you if you are no longer in charge*, they instead will gain respect for you as a facilitator, as their learning becomes centered around their own interests. Student-led inquiry can help you better include students who are not readily engaged by classroom work or who frequently experience behavioral challenges.

## **14.3 Starting Small and Dreaming Big**

Place-based scientific inquiry outside is very different from traditional classroom instruction. Making the transition is nontrivial and takes time. Give yourself the grace of starting small and dreaming big.

Small steps also build your confidence and promote your learning. You may find it takes a few trial runs to develop the necessary facilitation and group management skills and to get familiar with the flow of a project within your time constraints. Start with a relatively short project with limited scope.

Small steps and small inquiry projects will help get your students ready for larger successes later on. Once your students become familiar with open-ended inquiry and see you as the facilitator, you will find it possible to incorporate similar approaches into other projects or lessons you are already doing in the classroom. For example, labs where students are meant to get a fixed result can be replaced by ones where the students do not know the outcome or can choose their own methods.

  You are developing a group of students who are ready to learn for themselves and who are ready to see you as a partner in their learning. You are moving toward a more student-centered and justicefocused classroom. You are working to dismantle power structures that hold students back from the successes they deserve. You are building relationships and community. You are connecting people to places and working toward healing.

You are at the end of this book, and the beginning of your experience.

# **References**

256

260

# **Index**

Notes: *Italicised* page numbers refer to figures and **bold** refers to tables in the text.

accessibility 38, 68 access to additional supplies 66 acknowledging 133; different skills 105; resolving conflicts and 119 additional supplies, access to 66 alienated 111 allowing all voices to be heard 104–105 allowing students to discuss in pairs 100 American Sign Language (ASL) 50, *52*, *56* analysis of variance (ANOVA) 213, *216* analyzing and sense-making 198–217; drawing conclusions from data 198–204; statistical tests 212–217; visually interpreting results 205–211 anchor chart 138; blank 157; class whiteboard 158; generic *157*; hypotheses component of 156; with study design predetermined by the teacher *159*; verbal or graphical predictions on 199; *see also* designing a study and using an anchor chart animals: behavior 201; noises 121; place use 80; questions 146

answering questions with more questions 100 artists/illustrator 180, *182* asking general questions 100 asking probing questions 100 asking process questions 100 asking questions that do not have yes/ no answers 100 asking specific time-centered questions 100 assessing learning 243–251; student-centered reflection and feedback 247–251; why (or why not) to assess 243–246 auditory equipment 81; *see also* equipment and data collection at low cost avoiding stereotypes 43

background information, acquiring 87–90; concepts 87; elementary (3–5) 88; equity and inclusion 87; examples 88, *89*, 90; grade-level differentiation 88; high (9–12) 88; implementation 87–88; middle (6–8) 88 bad handwriting 187

barriers minimizing 43 basic needs 66 being comfortable with some chaos 130 blindfolded: search 121; student 82, *84*, 187 blind/low vision students 50–51; accessibility 38, 54, 65, 68, 238; colorblind 205; technology 9, 13, 51, 178 boundaries 67 broader community 239 building a strong question 141–147; concepts 141–142, **142**–**143**; elementary (3–5) 145; equity and inclusion 144; examples 146, *147*; grade-level differentiation 145–146; high (9–12) 146; implementation 144–145; middle (6–8) 146

centering student identity 10–12 chaos, being comfortable 130 check-ins, having regular 130 circle observations 82 classroom 243 close observation activity 138 close observation drawing 80 codes 187 collecting data 185–192; concepts 185–186; elementary (3–5) 188; examples *188*–*192*; grade-level differentiation 188; implementation 186–188 collecting data outdoors 183–197; collecting data 185–192; practicing collecting data 183–185; securing data 192–197 colonization 15–16 color rainbow 82 communicating expectations 66 communicating schedules 66

121–122 communication tools 67 community event 237–242; concepts 237; elementary (3–5) 240; equity and inclusion 238; examples *240*– *242*; grade-level differentiation 240; high (9–12) 240; implementation 238–239; middle (6–8) 240 concepts: acquiring background information 87; acquiring equipment and collecting data at low cost 58; building a strong question 141–142, **142**–**143**; choosing a topic 24; choosing how much to measure 172–173; choosing roles 179; choosing what to do (protocols, checklists, and datasheets) 176; collecting data 185–186; cost considerations 35; designing a study and using an anchor chart 147–156 *148*; dividing students into groups 73; drawing conclusions from data 198–200; encouraging positive behavior from groups 113–114; encouraging positive behavior from individuals 107; exploration, observation, and sense of place 79; facilitating question development 136; getting groups back on track 132; handling multiple groups 129; holding a community event 237; how and why to share projects 226; identifying multiple hypotheses and predictions, or not 162–165; partnering with volunteers 53; place considerations 38; practicing collecting data 183; recognition 223; recognizing relationships and responsibility to place 90; reflection

218–219; role as teacher 20;

communication skills, building of

safety and logistical preparation 65; scaffolding projects into curriculum 32; scheduling and time considerations 26–27; securing data 192; setting behavioral expectations 102–103; small-group facilitation 97; statistical tests **212**–**213**, 212–214; student-centered reflection and feedback 247; student identity considerations 42; supporting presentation development 232; team building 118–119; team contracts and check-ins 125; universal design, special needs, and disability 46; using notebooks 75; visually interpreting results 205; why (or why not) to assess 243–244 consciousness critical 3–5, 7, 9, 19, 42 constructivism, place-based teaching 8 content linkages 38 contrasts 38 conversation on group dynamics 133 cost considerations 35–37; categories 35; concepts 35; elementary (3–5) 36; equity and inclusion 35; examples *37*; grade-level differentiation 36; high (9–12) 36; implementation 36; middle (6–8) 36 Crosscutting Concepts 218; scaffolding projects into curriculum 32, 33; scheduling and time considerations 30 culture/cultural/culturally 12, 232; artifacts 40; differences 43; erasure of 15; expectations 132; friendships and *246*; identities 12; Indigenous science 18; informed 44; meaningful places *41*; of mutual respect 27; responsive 17; supportive 43; sustaining 17; trespassing on significant sites 40; values 7, 12; *see also* white supremacy

cups/envelopes/sealable plastic bags 59 curiosity and exploration 103 curriculum *see* scaffolding projects into curriculum

data collection *see* collecting data outdoors; planning data collection data recorder 180, *181* data recording techniques 187 day-in-the-life 82 decolonization: discomfort 17; placebased outdoor teaching 17; place relationships and 219; teaching 15–17, 18 designing a study and using an anchor chart 147–162; concepts 147–156 *148*; elementary (3–5) 158; examples *159*–*160*, 161–162; grade-level differentiation 158; high (9–12) 158; implementation 156–158, *157*; middle (6–8) 158 destroying things 110 differences from home 82 digital team contract *128* disability 22; additional support for 46; identity-related biases 50; in outdoor activities 17; student identities 21, 46; students with physical 50; *see also* universal design, special needs, and disability Disciplinary Core Ideas, NGSS 13, 14, *26*, 218; scaffolding projects into curriculum 32, 33; scheduling and time considerations 30

discrimination, marginalization and 11 disengaging from the group 109

dividing students into groups 73–75; concepts 73; elementary (3–5) 74; equity and inclusion 73; examples *75*; grade-level differentiation 74; high (9–12) 74; implementation 74; middle (6–8) 74 doing nothing 133 dominating/directing others 109 drawing conclusions from data 198–204; concepts 198–200; elementary (3–5) 201–202; equity and inclusion 200; examples *202*–*204*; grade-level differentiation 201–202; high (9–12) 202; implementation 201; middle (6–8) 202 dreaming big 254–255 durability 38

education, empowering young people *4*

elementary school (3–5): acquiring background information 88; building a strong question 145; choosing a topic 25; choosing how much to measure 174; choosing what to do (protocols, checklists, and datasheets) 178; collecting data 188; cost considerations 36; designing a study and using an anchor chart 158; dividing students into groups 74; drawing conclusions from data 201– 202; encouraging positive behavior from groups 116; facilitating question development 138; getting groups back on track 134; handling multiple groups 130; holding a community event 240; how and why to share projects 228; identifying multiple hypotheses and predictions, or not 169; partnering with

volunteers 55; place considerations 40; practicing collecting data 184; recognition 224; reflection 221; role as teacher 22; safety and logistical preparation 69; scaffolding projects into curriculum 33; scheduling and time considerations 29; securing data 194; setting behavioral expectations 105; statistical tests 214–215; student-centered reflection and feedback 249; student identity considerations 43–44; supporting presentation development 234; team building 123; team contracts and check-ins 127; using notebooks 76; visually interpreting results 207; why (or why not) to assess 245 English language learners 50 equipment 68; auditory 81; keeper 180; misuse 187; tactile 81–82; visual 80–81; writing 82 equipment and data collection at: concepts 58; equity and inclusion 59; examples *61*–*64*; grade-level differentiation 60; high (9–12) 60; implementation 59–60; low cost 58–64

equity and inclusion: acquiring background information 87; acquiring equipment and collecting data at low cost 59; building a strong question 144; choosing a topic 24; choosing roles 180; choosing what to do (protocols, checklists, and datasheets) 177; cost considerations 35; dividing students into groups 73; drawing conclusions from data 200; encouraging positive behavior from groups 114; encouraging positive behavior from individuals 107; exploration, observation,

and sense of place 79; facilitating question development 136–137; getting groups back on track 132; handling multiple groups 129; holding a community event 238; how and why to share projects 227; identifying multiple hypotheses and predictions, or not 165; partnering with volunteers 53–54; place considerations 39; practicing collecting data 183; recognition 223; recognizing relationships and responsibility to place 90–91; reflection 219; role as teacher 20; safety and logistical preparation 65–66; scheduling and time considerations 27; setting behavioral expectations 103; small-group facilitation 97–98; statistical tests 214; student-centered reflection and feedback 247; student identity considerations 42; supporting presentation development 232; team building 119; team contracts and check-ins 126; universal design, special needs, and disability 46–47; using notebooks 76; visually interpreting results 205–206; why (or why not) to assess 244–245 ethics 38; building a strong question 141; co-discovery journey with students 91; hypothesis-testing study design 148; protocol 177; respect and 92; information 88, *89*, 90; acquiring equipment and collecting data at low cost *61*–*64*; building a strong question 146, *147*; choosing a topic *26*; choosing how much to measure *175*–*176*; choosing roles *181*–*182*; choosing what to do (protocols, checklists, and datasheets)

*179*; collecting data *188*–*192*; cost considerations *37*; designing a study and using an anchor chart *159*–*160*, 161–162; dividing students into groups *75*; drawing conclusions from data *202*–*204*; encouraging positive behavior from groups *117*–*118*; encouraging positive behavior from individuals *112*–*113*; exploration, observation, and sense of place *83*–*86*; facilitating question development 139, *140*–*141*; getting groups back on track *135*; handling multiple groups 131; holding a community event *240*–*242*; how and why to share projects *229*– *231*; identifying multiple hypotheses and predictions, or not *170*–*172*; partnering with volunteers *56*–*58*; place considerations *40*–*41*; practicing collecting data *184*–*185*; recognition *225*; recognizing relationships and responsibility to place *93*–*94*, 94–95, *95*–*96*; reflection *222*; role as teacher 22; safety and logistical preparation *69*–*72*; scaffolding projects into curriculum *34*; scheduling and time considerations 30, *30*–*31*; securing data *194*–*197*; setting behavioral expectations *106*–*107*; small-group facilitation *101*–*102*; statistical tests 215, *216*–*217*; student-centered reflection and feedback *249*–*251*; student identity considerations *44*–*45*; supporting presentation development *234*–*237*; team building *123*–*125*; team contracts and check-ins *127*– *128*; universal design, special needs, and disability *51*–*52*; using notebooks *77*–*78*; visually interpreting results *207*–*211*, 208–210; why (or why not) to assess *246*

expectations *see* scope and expectations exploration, observation, and sense of place 79–86; concepts 79; equity and inclusion 79; examples *83*–*86*; implementation 79–83 exploration and team building 73–96; acquiring background information 87–90; dividing students into groups 73–75; exploration, observation, and sense of place 79–86; recognizing relationships and responsibility to place 90–96; using notebooks 75–78 explorer 180 exploring the environment 108 expressing prejudices 110 extra supplies 68

facilitative question-based approach 98 facilities 67 factual correctness 100 feasible protocol 177 feeling afraid 110 feeling alienated 111 feeling uncomfortable 111 504 plan 48–49 five senses observations 137 five things in common 120 float between groups 129–130 forecasting 111 forest/lake area, question 139 free write 82 Freire, P. 3 Frye, M. 10

gender: identity-related biases 244; student being powerful or oppressed 10; student identity 4, 21 getting groups back on track 132–135; concepts 132; elementary (3–5) 134; equity and inclusion 132; examples

*135*; grade-level differentiation 134; implementation 133–134 getting to know names and each other 120–121

grade-level differentiation: acquiring background information 88; acquiring equipment and collecting data at low cost 60; building a strong question 145–146; choosing a topic 25; choosing how much to measure 174; choosing what to do (protocols, checklists, and datasheets) 178; collecting data 188; cost considerations 36; designing a study and using an anchor chart 158; dividing students into groups 74; drawing conclusions from data 201–202; encouraging positive behavior from groups 116; facilitating question development 138–139; getting groups back on track 134; handling multiple groups 130; holding a community event 240; how and why to share projects 228; identifying multiple hypotheses and predictions, or not 169; partnering with volunteers 55; place considerations 40; practicing collecting data 184; recognition 224–225; recognizing relationships and responsibility to place 92; reflection 221–222; role as teacher 22; safety and logistical preparation 69; scaffolding projects into curriculum 33; scheduling and time considerations 29–30; securing data 194; setting behavioral expectations 105; statistical tests 214–215; student-centered reflection and feedback 249; student identity considerations 43–44; supporting

presentation development 234; team building 123; team contracts and check-ins 127; using notebooks 76; visually interpreting results 207; why (or why not) to assess 245

group dynamics 27; conversation on 133; help the group 98; invest in team building 43; parents/guardians complicating 54; plan for flexibility 28; positive 112; self-regulation in 116; student engagement and 97; students self-awareness of 98; team building 115 guided poem 82 guidelines, establishing and

communicating 43

helping the group 108 high school (9–12): acquiring background information 88; acquiring equipment and collecting data at low cost 60; building a strong question 146; choosing a topic 25; choosing how much to measure 174; cost considerations 36; designing a study and using an anchor chart 158; dividing students into groups 74; drawing conclusions from data 202; encouraging positive behavior from groups 116; facilitating question development 139; handling multiple groups 130; holding a community event 240; how and why to share projects 228; identifying multiple hypotheses and predictions, or not 169; place considerations 40; recognition 225; reflection 222; role as teacher 22; safety and logistical preparation 69; scaffolding projects into curriculum 33; scheduling and time considerations

30; statistical tests 215; student identity considerations 44; team contracts and check-ins 127; visually interpreting results 207; why (or why not) to assess 245 how and why to share projects 226–231; concepts 226; elementary (3–5) 228; equity and inclusion 227; examples *229*–*231*; grade-level differentiation 228; high (9–12) 228; implementation 227–228; middle (6–8) 228 human development 244 human knot 121 human place use 80 hypotheses 162–164; *see also* identifying multiple hypotheses and predictions, or not hypothesis-driven projects 199, 205 hypothesis-testing study design 148

"I am like" poem 82 ice cube trays 59 identifying multiple hypotheses and predictions, or not 162–172; concepts 162–165; elementary (3–5) 169; equity and inclusion 165; examples *170*–*172*; grade-level differentiation 169; high (9–12) 169; implementation 165–168; middle (6–8) 169 identity *see* student identity

identity-based marginalization 11 implementation: acquiring background information 87–88; acquiring equipment and collecting data at low cost 59–60; building a strong question 144–145; choosing a topic 24–25; choosing how much to measure 173–174; choosing roles 180; choosing what

to do (protocols, checklists, and datasheets) 177–178; collecting data 186–188; cost considerations 36; designing a study and using an anchor chart 156–158, *157*; dividing students into groups 74; drawing conclusions from data 201; encouraging positive behavior from groups 115–116; encouraging positive behavior from individuals 107–112; exploration, observation, and sense of place 79–83; facilitating question development 137–138; getting groups back on track 133–134; handling multiple groups 129–130; holding a community event 238–239; how and why to share projects 227–228; identifying multiple hypotheses and predictions, or not 165–168; partnering with volunteers 54–55; place considerations 39–40; practicing collecting data 183–184; recognition 223–224; recognizing relationships and responsibility to place 91–92; reflection 219–221; role as teacher 21–22; safety and logistical preparation 66–68; scaffolding projects into curriculum 33; scheduling and time considerations 27–29; securing data 192–193; setting behavioral expectations 103–105; small-group facilitation 98–100; statistical tests 214; student-centered reflection and feedback 247–249; student identity considerations 42–43; supporting presentation development 232–233; team building 119–122; team contracts and check-ins 126; universal design, special needs, and

disability 47–51; using notebooks 76; visually interpreting results 206; why (or why not) to assess 245 improving behavior 108 inclusive protocol 177 Indigenous: name 91; peoples 1, 15, 17, 90, 92; perspectives 19; rights 38, 90, 92; science 18–19; students 7, 18, 19, 90, *93*; wilderness 17 Individualized Education Plan 27, 48–49 information 66–67; representation 47 inquiry-based teaching 7–8, 11; benefits of 8; perspective 18 interviews 81 introducing partner (paired activity) 120

Jones, K. 11 justice-first science teaching 3–4, 10

kitchen/luggage scale 59 kitchen thermometers 59 kitchen timers 59

land acknowledgment 92 land education, place-based inquiry linked to 8 laser eyes 122 lava lake 122 lava river 121–122, *124* Leave No Trace principles 67, 104 Leonardo, Z. 11 level 59 Leveraging Crosscutting Concepts, NGSS 13–14 linear regression 213–215, *216* logistics 38 lost equipment 186; *see also* equipment; equipment and data collection at low cost lost notebooks/pencils 187

magic stick 121, *123* magnifying lens/tweezers 59 marginalization: discrimination and 11; identity-based 11; role of teaching outdoors in combatting 11; within student groups 98 measure, choosing how to 172–176; concepts 172–173; elementary (3–5) 174; examples *175*–*176*; grade-level differentiation 174; high (9–12) 174; implementation 173–174; middle (6–8) 174 measuring tape/meter stick 59 medical: equipment 35; forms for information 66; situations 67; training 54; *see also* partnering with volunteers; safety and logistical preparation meditative/reflective 83 meet a tree 82, *84* mental breaks 119 mental map 83 mental picture 166 middle school (6–8): acquiring background information 88; building a strong question 146; choosing a topic 25; choosing how much to measure 174; choosing what to do (protocols, checklists, and datasheets) 178; designing a study and using an anchor chart 158; dividing students into groups 74; drawing conclusions from data 202; encouraging positive behavior from groups 116; facilitating question development 138–139; handling multiple groups 130; holding a community event 240; how and why to share projects 228; identifying multiple hypotheses and predictions, or not 169; place considerations

40; recognition 224; recognizing relationships and responsibility to place 92; reflection 222; role as teacher 22; safety and logistical preparation 69; scaffolding projects into curriculum 33; scheduling and time considerations 30; securing data 194; setting behavioral expectations 105; statistical tests 215; student identity considerations 44; supporting presentation development 234; team building 123; team contracts and check-ins 127; visually interpreting results 207 monster game 122, *125* morphonames 187, *192* motivation of students 4 multiple groups, handling 129–131; concepts 129; elementary (3–5) 130; equity and inclusion 129; examples 131; grade-level differentiation 130; high (9–12) 130; implementation 129–130; middle (6–8) 130 multiple hypotheses 142, 163, 199; *see also* identifying multiple hypotheses and predictions, or not

names, getting to know 120–121 natural art 81 natural history notes 81 neurodiverse students 49–50 Next Generation Science Standards (NGSS) 12–14, 243; learning as three-dimensional 13; learning beyond 14–15; student-led inquiry 32 Nez Perce *41*, *93* normalizing having needs 66 norms 102

notebooks, use of 75–78; concepts 75; elementary (3–5) 76; equity and inclusion 76; examples *77*–*78*; grade-level differentiation 76; implementation 76

observations, making 108 Okun, T. 11 open-ended questions 100; *see also* question and study design, developing of outcomes *see* sharing outcomes oven 59 overcoming common fears 253–254 oversharing opinions/facts 109

packing lists 68

paint sample booklet 59

paired drawing 81 paper damage 187

parents/guardians 239; care taken when recruiting 53; making group dynamics complicate 54; not to be solicited for funding student needs 59; time spent by community volunteer organizations in lieu of 53–54 partnering with volunteers 53–58; concepts 53; elementary (3–5) 55; equity and inclusion 53–54; examples *56*–*58*; grade-level differentiation 55; implementation 54–55 Pascua Yaqui 1, *44*

people, questions 146 permits 38 place-based inquiry 8, 12, 13, 20, 21, 32 place-based outdoor projects 244

place-based outdoor teaching: decolonization 17; role in students' awareness 17

place-based projects 24, 227

place-based scientific inquiry 8, 252; decolonizing knowledge 16; help to removes barriers and norms common to classrooms 11; *see also* Next Generation Science Standards (NGSS)

place-based teaching 4–7, 10; as foundation for integrating justice for disabled students 11; perspective of knowledge 18

place considerations 38–41; concepts 38; elementary (3–5) 40; equity and inclusion 39; examples *40*–*41*; grade-level differentiation 40; high (9–12) 40; implementation 39–40; middle (6–8) 40

place knowledge 43

placelessness, feelings of 6

planning data collection 172–182; choosing how much to measure 172–176; choosing roles 179–182; choosing what to do (protocols, checklists, and datasheets) 176–179

plants, questions 146 plastic vials 59 positive behavior from groups, encouraging 113–118; concepts 113–114; elementary (3–5) 116; equity and inclusion 114; examples *117*–*118*; grade-level differentiation 116; high (9–12) 116; implementation 115–116; middle (6–8) 116 positive behavior from individuals, encouraging 107–113; concepts

107; equity and inclusion 107; examples *112*–*113*; implementation 107–112 positive relationships, building 122

potentially marginalized students 49 practicing collecting data 183–185; concepts 183; elementary (3–5) 184; equity and inclusion 183; examples *184*–*185*; grade-level differentiation 184; implementation 183–184 praising 112 predator-prey 122, *124* prejudices 110 pristineness/pristine 6, 11, 39, 90; *see also* wilderness/wild proximity 111 psychosocial 243 public 67–68 PVC piping 59

quadrat 187

question and study design, developing of 136–172; building a strong question 141–147; designing a study and using an anchor chart 147–162; facilitating question development 136–141; identifying multiple hypotheses and predictions, or not 162–172

question development, facilitating 136–141; concepts 136; elementary (3–5) 138; equity and inclusion 136–137; examples 139, *140*–*141*; grade-level differentiation 138–139; high (9–12) 139; implementation 137–138; middle (6–8) 138–139 question generation 137–138 question templates 138 question types **142**–**143**

race: identity-related biases 244; student being powerful or oppressed 10; student identity 4, 21

randomization 187 recognition 223–225; concepts 223; elementary (3–5) 224; equity and inclusion 223; examples *225*; grade-level differentiation 224–225; high (9–12) 225; implementation 223–224; middle (6–8) 224 redirecting attention 111 reflecting and recognizing success 218–225; recognition 223–225; reflection 218–222 reflection 218–222; around findings 220; around justice 221; around place relationships 221; around process 220; around self/peers 220; concepts 218–219; elementary (3–5) 221; equity and inclusion 219; examples *222*; grade-level differentiation 221–222; high (9–12) 222; implementation 219–221; middle (6–8) 222 relationships 5, 83 relationships and responsibility to place 90–96; concepts 90; equity and inclusion 90–91; examples *93*–*94*, 94–95, *95*–*96*; grade-level differentiation 92; implementation 91–92; middle (6–8) 92 Relph, E. 5 relying on other adults 130 removal/removing students 112 resonance and meaning 38 resources and planning safe logistics 53–72; acquiring equipment and collecting data at low cost 58–64; partnering with volunteers 53–58; safety and logistical preparation 65–72 respect for organisms 104 respect for others 104–105

respect for place 104

role as teacher 20–23; concepts 20; elementary (3–5) 22; equity and inclusion 20; examples 22; gradelevel differentiation 22; high (9–12) 22; implementation 21–22; middle (6–8) 22 roles selection 179–182; concepts 179; equity and inclusion 180; examples *181*–*182*; implementation 180 rope shape-making 121 rubric 233, 248 rules 102

safety and logistical preparation 38, 65–72; concepts 65; elementary (3–5) 69; equity and inclusion 65–66; examples *69*–*72*; grade-level differentiation 69; high (9–12) 69; implementation 66–68; middle (6–8) 69 sampling techniques 187–188 scaffolding projects into curriculum 32–34; concepts 32; elementary (3–5) 33; examples *34*; grade-level differentiation 33; high (9–12) 33; implementation 33; middle (6–8) 33 scheduling and time considerations: concepts 26–27; elementary (3–5) 29; equity and inclusion 27; examples 30, *30*–*31*; grade-level differentiation 29–30; high (9–12) 30; implementation 27–29; middle (6–8) 30 school community members 238–239 schoolyard trash production, studying *12* Science and Engineering Practices 13, 218; scaffolding projects into curriculum 32; scheduling and time considerations 30 scope and expectations 20–52; choosing a topic 24–31; cost considerations

35–37; place considerations 38–41; role as teacher 20–23; scaffolding projects into curriculum 32–34; student identity considerations 42–45; universal design, special needs, and disability 46–52 securing data 192–197; concepts 192; elementary (3–5) 194; examples *194*–*197*; grade-level differentiation 194; implementation 192–193; middle (6–8) 194 sense-making *see* analyzing and sense-making sense of place 28, 48, 79–86; *see also*

exploration, observation, and sense of place

setting behavioral expectations 102– 107; concepts 102–103; elementary (3–5) 105; equity and inclusion 103; examples *106*–*107*; grade-level differentiation 105; implementation 103–105; middle (6–8) 105 sharing and reviewing 133 sharing outcomes 226–242; holding a community event 237–242; how and why to share projects 226–231; supporting presentation development 232–237

show and tell 138

signaling 112

silence 83

simplifying 133

small-group facilitation 97–102; concepts 97; equity and inclusion 97–98; examples *101*–*102*; implementation 98–100 soliciting multiple answers 100 solving problems 108 sound inventory 81 sound map 81

starting small 254–255

statistical tests 212–217; concepts **212**–**213**, 212–214; elementary (3–5) 214–215; equity and inclusion 214; examples 215, *216*–*217*; grade-level differentiation 214–215; high (9–12) 215; implementation 214; middle (6–8) 215 statistics 33; descriptive 198, 205, 214; inferential 200, 212; summary 207 stereotypes 43, 54, 79, 98 string/rope 59 strong inference 163, 164, 199, 232 student(s): action and expression 47–48; behaviors 108–111; with disabilities 50; engagement 48; learning *5*; motivation of 4; teams 238; view 252–253 student-centered reflection and feedback 247–251; concepts 247; elementary (3–5) 249; equity and inclusion 247; examples *249*–*251*; grade-level differentiation 249; implementation 247–249 student-centered teaching 10, 97 student identity *10*, 10–12, 42–45; concepts 42; elementary (3–5) 43–44; equity and inclusion 42; examples *44*–*45*; grade-level differentiation 43–44; high (9–12) 44; implementation 42–43; middle (6–8) 44 study design 14, 30; anchor chart with *159*; creative 15; cross-sectional 215; *see also* question and study design, developing of suburban schoolyard, question 139 summarizing 133 supporting presentation development 232–237; concepts 232; elementary (3–5) 234; equity and inclusion 232;

examples *234*–*237*; grade-level differentiation 234; implementation 232–233; middle (6–8) 234

tactile equipment 81–82 tactile exploration 81 taking a break 133, *135* taking care of one another's needs 105 taking immediate action 133 task-setting 111 teachers view 252–253 teaching outside 8–10, *9*; combat marginalization of disabled students 11; schoolyard or remote sites 65 team building 43, 118–125; concepts 118–119; elementary (3–5) 123; equity and inclusion 119; examples *123*–*125*; grade-level differentiation 123; implementation 119–122; middle (6–8) 123; *see also* exploration and team building team contracts and check-ins 125–128; concepts 125; elementary (3–5) 127; equity and inclusion 126; examples *127*–*128*; grade-level differentiation 127; high (9–12) 127; implementation 126; middle (6–8) 127

teams and conflict resolving 97–135; encouraging positive behavior from groups 113–118; encouraging positive behavior from individuals 107–113; getting groups back on track 132–135; handling multiple groups 129–131; setting behavioral expectations 102–107; small-group facilitation 97–102; team building 118–125; team contracts and checkins 125–128

ten minutes of silence activity 137 tick marks 187

time and schedule 105 timer 180 timetable for day's work 130 timing activity chunks 111 Tohono O'odham 1 tools 138, 254; communication 67; scarcity 58; for speaking and listening 115; statistical tests 200; usage 108 topic selection 24–31; concepts 24; elementary (3–5) 25; equity and inclusion 24; examples *26*; gradelevel differentiation 25; high (9–12) 25; implementation 24–25; middle (6–8) 25 transect 187, *191* transportation mode 68 trowels 59 trust fall and pass-around 122 t-test 213–214, 215, *216*–*217* twenty questions activity 137

ungrading 247

universal design, special needs, and disability 46–52; concepts 46; equity and inclusion 46–47; examples *51*–*52*; implementation 47–51; *see also* disability Universal Design for Learning (UDL) 46 urban schoolyard, question 139

values 5, 7, 9, 12, 16, 42, 125, 136, 205, 237 valuing student suggestions 100 visual equipment 80–81 visually interpreting results 205–211; concepts 205; elementary (3–5) 207; equity and inclusion 205–206; examples *207*–*211*, 208–210; gradelevel differentiation 207; high (9–12)

207; implementation 206; middle (6–8) 207 volunteers *see* partnering with volunteers

wait times 55, 100 wander 109–110 weather 68; animal behavior and 201; communicating expectations 66; effects of 14; inclement 76; learning to use digital meter *179*; study of natural world *31* welfare checker 180 what to do (protocols, checklists, and datasheets) 176–179; concepts 176; elementary (3–5) 178; equity and inclusion 177; examples *179*; grade-level differentiation 178; implementation 177–178; middle (6–8) 178 white supremacy culture: discouraging conflict in favor of absolute viewpoint 114; inquiry-based teaching 11 why (or why not) to assess 243–246; concepts 243–244; elementary (3–5) 245; equity and inclusion 244–245; examples *246*; grade-level differentiation 245; high (9–12) 245; implementation 245 wilderness/wild 6, 17, 30; *see also* pristineness/pristine withdrawing 110 writing 82; assignments 32; blog post for Internet distribution 231; drawing skills and 234; implementing a risk management plan and 65; natural

young people, education empowering *4*

138

history notes *85*; short observations